Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function.

Human T cells are central effectors of immunity and cancer immunotherapy. CRISPR-based functional studies in T cells could prioritize novel targets for drug development and improve the design of genetically reprogrammed cell-based therapies. However, large-scale CRISPR screens have been challenging in primary human cells. We developed a new method, single guide RNA (sgRNA) lentiviral infection with Cas9 protein electroporation (SLICE), to identify regulators of stimulation responses in primary human T cells. Genome-wide loss-of-function screens identified essential T cell receptor signaling components and genes that negatively tune proliferation following stimulation. Targeted ablation of individual candidate genes characterized hits and identified perturbations that enhanced cancer cell killing. SLICE coupled with single-cell RNA sequencing (RNA-seq) revealed signature stimulation-response gene programs altered by key genetic perturbations. SLICE genome-wide screening was also adaptable to identify mediators of immunosuppression, revealing genes controlling responses to adenosine signaling. The SLICE platform enables unbiased discovery and characterization of functional gene targets in primary cells.

RASA2 ablation in T cells boosts antigen sensitivity and long-term function.

The efficacy of adoptive T cell therapies for cancer treatment can be limited by suppressive signals from both extrinsic factors and intrinsic inhibitory checkpoints1,2. Targeted gene editing has the potential to overcome these limitations and enhance T cell therapeutic function3-10. Here we performed multiple genome-wide CRISPR knock-out screens under different immunosuppressive conditions to identify genes that can be targeted to prevent T cell dysfunction. These screens converged on RASA2, a RAS GTPase-activating protein (RasGAP) that we identify as a signalling checkpoint in human T cells, which is downregulated upon acute T cell receptor stimulation and can increase gradually with chronic antigen exposure. RASA2 ablation enhanced MAPK signalling and chimeric antigen receptor (CAR) T cell cytolytic activity in response to target antigen. Repeated tumour antigen stimulations in vitro revealed that RASA2-deficient T cells show increased activation, cytokine production and metabolic activity compared with control cells, and show a marked advantage in persistent cancer cell killing. RASA2-knockout CAR T cells had a competitive fitness advantage over control cells in the bone marrow in a mouse model of leukaemia. Ablation of RASA2 in multiple preclinical models of T cell receptor and CAR T cell therapies prolonged survival in mice xenografted with either liquid or solid tumours. Together, our findings highlight RASA2 as a promising target to enhance both persistence and effector function in T cell therapies for cancer treatment.

Genetic interactions in cancer progression and treatment.

As cancer cell genomes are unveiled at a breathtaking pace, the genetic principles at play in cancer are emerging in all their complexity, prompting the assessment of classical genetic interaction models. Here, we discuss the implications of these findings for cancer progression and heterogeneity and for the development of new therapeutic approaches.

Iterative computational design and crystallographic screening identifies potent inhibitors targeting the Nsp3 macrodomain of SARS-CoV-2.

The nonstructural protein 3 (NSP3) of the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) contains a conserved macrodomain enzyme (Mac1) that is critical for pathogenesis and lethality. While small-molecule inhibitors of Mac1 have great therapeutic potential, at the outset of the COVID-19 pandemic, there were no well-validated inhibitors for this protein nor, indeed, the macrodomain enzyme family, making this target a pharmacological orphan. Here, we report the structure-based discovery and development of several different chemical scaffolds exhibiting low- to sub-micromolar affinity for Mac1 through iterations of computer-aided design, structural characterization by ultra-high-resolution protein crystallography, and binding evaluation. Potent scaffolds were designed with in silico fragment linkage and by ultra-large library docking of over 450 million molecules. Both techniques leverage the computational exploration of tangible chemical space and are applicable to other pharmacological orphans. Overall, 160 ligands in 119 different scaffolds were discovered, and 153 Mac1-ligand complex crystal structures were determined, typically to 1 Å resolution or better. Our analyses discovered selective and cell-permeable molecules, unexpected ligand-mediated conformational changes within the active site, and key inhibitor motifs that will template future drug development against Mac1.

A single inactivating amino acid change in the SARS-CoV-2 NSP3 Mac1 domain attenuates viral replication in vivo.

Despite unprecedented efforts, our therapeutic arsenal against SARS-CoV-2 remains limited. The conserved macrodomain 1 (Mac1) in NSP3 is an enzyme exhibiting ADP-ribosylhydrolase activity and a possible drug target. To determine the role of Mac1 catalytic activity in viral replication, we generated recombinant viruses and replicons encoding a catalytically inactive NSP3 Mac1 domain by mutating a critical asparagine in the active site. While substitution to alanine (N40A) reduced catalytic activity by ~10-fold, mutations to aspartic acid (N40D) reduced activity by ~100-fold relative to wild-type. Importantly, the N40A mutation rendered Mac1 unstable in vitro and lowered expression levels in bacterial and mammalian cells. When incorporated into SARS-CoV-2 molecular clones, the N40D mutant only modestly affected viral fitness in immortalized cell lines, but reduced viral replication in human airway organoids by 10-fold. In mice, the N40D mutant replicated at >1000-fold lower levels compared to the wild-type virus while inducing a robust interferon response; all animals infected with the mutant virus survived infection. Our data validate the critical role of SARS-CoV-2 NSP3 Mac1 catalytic activity in viral replication and as a promising therapeutic target to develop antivirals.

TEAD Proteins Associate With DNA Repair Proteins to Facilitate Cellular Recovery From DNA Damage.

Transcriptional enhanced associate domain family members 1 to 4 (TEADs) are a family of four transcription factors and the major transcriptional effectors of the Hippo pathway. In order to activate transcription, TEADs rely on interactions with other proteins, such as the transcriptional effectors Yes-associated protein and transcriptional co-activator with PDZ-binding motif. Nuclear protein interactions involving TEADs influence the transcriptional regulation of genes involved in cell growth, tissue homeostasis, and tumorigenesis. Clearly, protein interactions for TEADs are functionally important, but the full repertoire of TEAD interaction partners remains unknown. Here, we employed an affinity purification mass spectrometry approach to identify nuclear interacting partners of TEADs. We performed affinity purification mass spectrometry experiment in parallel in two different cell types and compared a wildtype TEAD bait protein to a nuclear localization sequence mutant that does not localize to the nucleus. We quantified the results using SAINT analysis and found a significant enrichment of proteins linked to DNA damage including X-ray repair cross-complementing protein 5 (XRCC5), X-ray repair cross-complementing protein 6 (XRCC6), poly(ADP-ribose) polymerase 1 (PARP1), and Rap1-interacting factor 1 (RIF1). In cellular assays, we found that TEADs co-localize with DNA damage-induced nuclear foci marked by histone H2AX phosphorylated on S139 (γH2AX) and Rap1-interacting factor 1. We also found that depletion of TEAD proteins makes cells more susceptible to DNA damage by various agents and that depletion of TEADs promotes genomic instability. Additionally, depleting TEADs dampens the efficiency of DNA double-stranded break repair in reporter assays. Our results connect TEADs to DNA damage response processes, positioning DNA damage as an important avenue for further research of TEAD proteins.

Reprogramming human T cell function and specificity with non-viral genome targeting.

Decades of work have aimed to genetically reprogram T cells for therapeutic purposes1,2 using recombinant viral vectors, which do not target transgenes to specific genomic sites3,4. The need for viral vectors has slowed down research and clinical use as their manufacturing and testing is lengthy and expensive. Genome editing brought the promise of specific and efficient insertion of large transgenes into target cells using homology-directed repair5,6. Here we developed a CRISPR-Cas9 genome-targeting system that does not require viral vectors, allowing rapid and efficient insertion of large DNA sequences (greater than one kilobase) at specific sites in the genomes of primary human T cells, while preserving cell viability and function. This permits individual or multiplexed modification of endogenous genes. First, we applied this strategy to correct a pathogenic IL2RA mutation in cells from patients with monogenic autoimmune disease, and demonstrate improved signalling function. Second, we replaced the endogenous T cell receptor (TCR) locus with a new TCR that redirected T cells to a cancer antigen. The resulting TCR-engineered T cells specifically recognized tumour antigens and mounted productive anti-tumour cell responses in vitro and in vivo. Together, these studies provide preclinical evidence that non-viral genome targeting can enable rapid and flexible experimental manipulation and therapeutic engineering of primary human immune cells.

Hypoxia Is a Dominant Remodeler of the Effector T Cell Surface Proteome Relative to Activation and Regulatory T Cell Suppression.

Immunosuppressive factors in the tumor microenvironment (TME) impair T cell function and limit the antitumor immune response. T cell surface receptors and surface proteins that influence interactions and function in the TME are proven targets for cancer immunotherapy. However, how the entire surface proteome remodels in primary human T cells in response to specific suppressive factors in the TME remains to be broadly and systematically characterized. Here, using a reductionist cell culture approach with primary human T cells and stable isotopic labeling with amino acids in cell culture-based quantitative cell surface capture glycoproteomics, we examined how two immunosuppressive TME factors, regulatory T cells (Tregs) and hypoxia, globally affect the activated CD8+ surface proteome (surfaceome). Surprisingly, coculturing primary CD8+ T cells with Tregs only modestly affected the CD8+ surfaceome but did partially reverse activation-induced surfaceomic changes. In contrast, hypoxia drastically altered the CD8+ surfaceome in a manner consistent with both metabolic reprogramming and induction of an immunosuppressed state. The CD4+ T cell surfaceome similarly responded to hypoxia, revealing a common hypoxia-induced surface receptor program. Our surfaceomics findings suggest that hypoxic environments create a challenge for T cell activation. These studies provide global insight into how Tregs and hypoxia remodel the T cell surfaceome and we believe represent a valuable resource to inform future therapeutic efforts to enhance T cell function.

The cancer cell map initiative: defining the hallmark networks of cancer.

Progress in DNA sequencing has revealed the startling complexity of cancer genomes, which typically carry thousands of somatic mutations. However, it remains unclear which are the key driver mutations or dependencies in a given cancer and how these influence pathogenesis and response to therapy. Although tumors of similar types and clinical outcomes can have patterns of mutations that are strikingly different, it is becoming apparent that these mutations recurrently hijack the same hallmark molecular pathways and networks. For this reason, it is likely that successful interpretation of cancer genomes will require comprehensive knowledge of the molecular networks under selective pressure in oncogenesis. Here we announce the creation of a new effort, The Cancer Cell Map Initiative (CCMI), aimed at systematically detailing these complex interactions among cancer genes and how they differ between diseased and healthy states. We discuss recent progress that enables creation of these cancer cell maps across a range of tumor types and how they can be used to target networks disrupted in individual patients, significantly accelerating the development of precision medicine.

Targeting DNA Damage Response and Replication Stress in Pancreatic Cancer.

BACKGROUND & AIMS: Continuing recalcitrance to therapy cements pancreatic cancer (PC) as the most lethal malignancy, which is set to become the second leading cause of cancer death in our society. The study aim was to investigate the association between DNA damage response (DDR), replication stress, and novel therapeutic response in PC to develop a biomarker-driven therapeutic strategy targeting DDR and replication stress in PC. METHODS: We interrogated the transcriptome, genome, proteome, and functional characteristics of 61 novel PC patient-derived cell lines to define novel therapeutic strategies targeting DDR and replication stress. Validation was done in patient-derived xenografts and human PC organoids. RESULTS: Patient-derived cell lines faithfully recapitulate the epithelial component of pancreatic tumors, including previously described molecular subtypes. Biomarkers of DDR deficiency, including a novel signature of homologous recombination deficiency, cosegregates with response to platinum (P < .001) and PARP inhibitor therapy (P < .001) in vitro and in vivo. We generated a novel signature of replication stress that predicts response to ATR (P < .018) and WEE1 inhibitor (P < .029) treatment in both cell lines and human PC organoids. Replication stress was enriched in the squamous subtype of PC (P < .001) but was not associated with DDR deficiency. CONCLUSIONS: Replication stress and DDR deficiency are independent of each other, creating opportunities for therapy in DDR-proficient PC and after platinum therapy.

Correction to: Timing of pubertal stages and breast cancer risk: the Breakthrough Generations Study.

As a consequence of responding to colleagues who asked about the publication of the original article [1], the authors have determined that the data published in Table 4 of the paper are incorrect.

Parity reduces mammary repopulating activity but does not affect mammary stem cells defined as CD24 + CD29/CD49fhi in mice.

BACKGROUND: Breast cancer (BCa) mortality is decreasing with early detection and improvement in therapies. The incidence of BCa, however, continues to increase, particularly estrogen-receptor-positive (ER +) subtypes. One of the greatest modifiers of ER + BCa risk is childbearing (parity), with BCa risk halved in young multiparous mothers. Despite convincing epidemiological data, the biology that underpins this protection remains unclear. Parity-induced protection has been postulated to be due to a decrease in mammary stem cells (MaSCs); however, reports to date have provided conflicting data. METHODS: We have completed rigorous functional testing of repopulating activity in parous mice using unfractionated and MaSC (CD24midCD49fhi)-enriched populations. We also developed a novel serial transplant method to enable us to assess self-renewal of MaSC following pregnancy. Lastly, as each pregnancy confers additional BCa protection, we subjected mice to multiple rounds of pregnancy to assess whether additional pregnancies impact MaSC activity. RESULTS: Here, we report that while repopulating activity in the mammary gland is reduced by parity in the unfractionated gland, it is not due to a loss in the classically defined MaSC (CD24+CD49fhi) numbers or function. Self-renewal was unaffected by parity and additional rounds of pregnancy also did not lead to a decrease in MaSC activity. CONCLUSIONS: Our data show instead that parity impacts on the stem-like activity of cells outside the MaSC population.

An integrated molecular profile of endometrioid ovarian cancer.

OBJECTIVE: Endometrioid ovarian carcinoma (EOVC) is an uncommon subtype of epithelial ovarian carcinoma and its molecular characteristics have been incompletely described. Prior sequencing investigations have been limited to targeted gene panels. We performed whole-exome sequencing to build an unbiased genetic profile of molecular alterations in endometrioid ovarian tumors with a goal to better understand this disease in the context of epithelial ovarian cancer and endometrioid uterine cancers. METHODS: Whole-exome sequencing was performed on EOVC samples (n = 26) and matched normals (n = 15). Gene mutations, mutational signatures and copy number variations (CNVs) informed a multi-dimensional regression classifier allowing for comparison to endometrial carcinoma (UCEC) and high grade serous ovarian carcinoma (HGSC). RESULTS: EOVC has a distinct and heterogeneous genomic profile. Identified significantly mutated genes in EOVC (PTEN, CTNNB1, PIK3CA, KMT2D, KMT2B, PIK3R1, ARID1A and TP53) occurred at similar frequencies in UCEC. Hypermutation, resulting from both mismatch repair deficiency (MMRd) and POLE mutation, was observed in EOVC at a frequency similar to UCEC. Like UCEC, a subset of EOVC cases closely resembled HGSC, harboring TP53 mutations, homologous recombination deficiency (HRd) mutation signatures and widespread CNVs. A machine-learning classifier confirmed the heterogeneous composition of EOVC. Potential therapeutic targets were identified in 62% of EOVC cases. We validated our findings in an orthogonal clinical sequencing registry of EOVC cases. CONCLUSIONS: We identified that EOVC are a molecularly heterogeneous group of epithelial ovarian cancers with distinct mutational signatures. In an age of precision oncology, there is a pressing need to understand the unique molecular drivers in uncommon histologic subtypes to facilitate genomically driven oncologic treatments.

Venoarterial extracorporeal membrane oxygenation for postcardiotomy cardiogenic shock-A six-year service evaluation.

Only a small number of English hospitals provide postcardiotomy venoarterial extracorporeal membrane oxygenation (VA-ECMO) and there are doubts about its efficacy and safety. The aim of this service evaluation was to determine local survival rates and report on patient demographics. This was a retrospective service evaluation of prospectively recorded routine clinical data from a tertiary cardiothoracic center in the United Kingdom offering services including cardiac and thoracic surgery, heart and lung transplantation, venovenous extracorporeal membrane oxygenation (VV-ECMO) for respiratory failure, and all types of mechanical circulatory support. In six years, 39 patients were supported with VA-ECMO for refractory postcardiotomy cardiogenic shock (PCCS). We analyzed survival data and looked for associations between survival rates and patient characteristics. The intervention was venoarterial-ECMO in patients with PCCS either following weaning from cardiopulmonary bypass or following a trial of inotropes and intra-aortic balloon counterpulsation on the intensive care unit. 30-day, hospital discharge, 1-year and 2-year survivals were 51.3%, 41%, 37.5%, and 38.5%, respectively. The median (IQR [range]) duration of support was 6 (4-9 [1-35]) days. Nonsurvival was associated with advanced age, shorter intensive care length of stay, and the requirement for postoperative hemofiltration. Reasonable survival rates can be achieved in selected patients who may have been expected to have a worse mortality without VA-ECMO. We suggest postoperative VA-ECMO should be available to all patients undergoing cardiac surgery be it in their own center or through an established pathway to a specialist center.

Klotho: A Major Shareholder in Vascular Aging Enterprises.

Accelerated vascular aging is a condition that occurs as a complication of several highly prevalent inflammatory conditions such as chronic kidney disease, cancer, HIV infection and diabetes. Age-associated vascular alterations underlie a continuum of expression toward clinically overt cardiovascular disease. This has contributed to the striking epidemiologic transition whereby such noncommunicable diseases have taken center stage as modern-day global epidemics and public health problems. The identification of α-Klotho, a remarkable protein that confers powerful anti-aging properties has stimulated significant interest. In fact, emerging data have provided fundamental rationale for Klotho-based therapeutic intervention for vascular diseases and multiple other potential indications. However, the application of such discoveries in Klotho research remains fragmented due to significant gaps in our molecular understanding of Klotho biology, as well as hurdles in clinical research and experimental barriers that must first be overcome. These advances will be critical to establish the scientific platform from which future Klotho-based interventional trials and therapeutic enterprises can be successfully launched.

Mapping genetic vulnerabilities reveals BTK as a novel therapeutic target in oesophageal cancer.

OBJECTIVE: Oesophageal cancer is the seventh most common cause of cancer-related death worldwide. Disease relapse is frequent and treatment options are limited. DESIGN: To identify new biomarker-defined therapeutic approaches for patients with oesophageal cancer, we integrated the genomic profiles of 17 oesophageal tumour-derived cell lines with drug sensitivity data from small molecule inhibitor profiling, identifying drug sensitivity effects associated with cancer driver gene alterations. We also interrogated recently described RNA interference screen data for these tumour cell lines to identify candidate genetic dependencies or vulnerabilities that could be exploited as therapeutic targets. RESULTS: By integrating the genomic features of oesophageal tumour cell lines with siRNA and drug screening data, we identified a series of candidate targets in oesophageal cancer, including a sensitivity to inhibition of the kinase BTK in MYC amplified oesophageal tumour cell lines. We found that this genetic dependency could be elicited with the clinical BTK/ERBB2 kinase inhibitor, ibrutinib. In both MYC and ERBB2 amplified tumour cells, ibrutinib downregulated ERK-mediated signal transduction, cMYC Ser-62 phosphorylation and levels of MYC protein, and elicited G1 cell cycle arrest and apoptosis, suggesting that this drug could be used to treat biomarker-selected groups of patients with oesophageal cancer. CONCLUSIONS: BTK represents a novel candidate therapeutic target in oesophageal cancer that can be targeted with ibrutinib. On the basis of this work, a proof-of-concept phase II clinical trial evaluating the efficacy of ibrutinib in patients with MYC and/or ERBB2 amplified advanced oesophageal cancer is currently underway (NCT02884453). TRIAL REGISTRATION NUMBER: NCT02884453; Pre-results.

Breast cancer risk in relation to history of preeclampsia and hyperemesis gravidarum: Prospective analysis in the Generations Study.

Preeclampsia and hyperemesis gravidarum are pregnancy complications associated with altered sex hormone levels. Previous studies suggest preeclampsia may be associated with a decreased risk of subsequent breast cancer and hyperemesis with an increased risk, but the evidence remains unclear. We used data from the Generations Study, a large prospective study of women in the United Kingdom, to estimate relative risks of breast cancer in relation to a history of preeclampsia and hyperemesis using Cox regression adjusting for known breast cancer risk factors. During 7.5 years average follow-up of 82,053 parous women, 1,969 were diagnosed with invasive or in situ breast cancer. Women who had experienced preeclampsia during pregnancy had a significantly decreased risk of premenopausal breast cancer (hazard ratio (HR) =0.67, 95% confidence interval (CI): 0.49-0.90) and of HER2-enriched tumours (HR = 0.33, 95% CI: 0.12-0.91), but there was no association with overall (HR = 0.90, 95% CI: 0.80-1.02) or postmenopausal (HR = 0.97, 95% CI: 0.85-1.12) breast cancer risk. Risk reductions among premenopausal women were strongest within 20 years since the last pregnancy with preeclampsia. Hyperemesis was associated with a significantly increased risk of HER2-enriched tumours (HR = 1.76, 95% CI: 1.07-2.87), but not with other intrinsic subtypes or breast cancer risk overall. These results provide evidence that preeclampsia is associated with a decreased risk of premenopausal and HER2-enriched breast cancer and that hyperemesis, although not associated with breast cancer risk overall, may be associated with raised risk of HER2-enriched tumours.

Domestic light at night and breast cancer risk: a prospective analysis of 105 000 UK women in the Generations Study.

BACKGROUND: Circadian disruption caused by exposure to light at night (LAN) has been proposed as a risk factor for breast cancer and a reason for secular increases in incidence. Studies to date have largely been ecological or case-control in design and findings have been mixed. METHODS: We investigated the relationship between LAN and breast cancer risk in the UK Generations Study. Bedroom light levels and sleeping patterns at age 20 and at study recruitment were obtained by questionnaire. Analyses were conducted on 105 866 participants with no prior history of breast cancer. During an average of 6.1 years of follow-up, 1775 cases of breast cancer were diagnosed. Cox proportional hazard models were used to calculate hazard ratios (HRs), adjusting for potential confounding factors. RESULTS: There was no association between LAN level and breast cancer risk overall (highest compared with lowest LAN level at recruitment: HR=1.01, 95% confidence interval (CI): 0.88-1.15), or for invasive (HR=0.98, 95% CI: 0.85-1.13) or in situ (HR=0.96, 95% CI: 0.83-1.11) breast cancer, or oestrogen-receptor (ER) positive (HR=0.98, 95% CI: 0.84-1.14); or negative (HR=1.16, 95% CI: 0.82-1.65) tumours separately. The findings did not differ by menopausal status. Adjusting for sleep duration, sleeping at unusual times (non-peak sleep) and history of night work did not affect the results. Night waking with exposure to light, occurring around age 20, was associated with a reduced risk of premenopausal breast cancer (HR for breast cancer overall=0.74, 95% CI: 0.55-0.99; HR for ER-positive breast cancer=0.69, 95% CI: 0.49-0.97). CONCLUSIONS: In this prospective cohort analysis of LAN, there was no evidence that LAN exposure increased the risk of subsequent breast cancer, although the suggestion of a lower breast cancer risk in pre-menopausal women with a history of night waking in their twenties may warrant further investigation.

DNA-Repair Defects and Olaparib in Metastatic Prostate Cancer.

BACKGROUND: Prostate cancer is a heterogeneous disease, but current treatments are not based on molecular stratification. We hypothesized that metastatic, castration-resistant prostate cancers with DNA-repair defects would respond to poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) inhibition with olaparib. METHODS: We conducted a phase 2 trial in which patients with metastatic, castration-resistant prostate cancer were treated with olaparib tablets at a dose of 400 mg twice a day. The primary end point was the response rate, defined either as an objective response according to Response Evaluation Criteria in Solid Tumors, version 1.1, or as a reduction of at least 50% in the prostate-specific antigen level or a confirmed reduction in the circulating tumor-cell count from 5 or more cells per 7.5 ml of blood to less than 5 cells per 7.5 ml. Targeted next-generation sequencing, exome and transcriptome analysis, and digital polymerase-chain-reaction testing were performed on samples from mandated tumor biopsies. RESULTS: Overall, 50 patients were enrolled; all had received prior treatment with docetaxel, 49 (98%) had received abiraterone or enzalutamide, and 29 (58%) had received cabazitaxel. Sixteen of 49 patients who could be evaluated had a response (33%; 95% confidence interval, 20 to 48), with 12 patients receiving the study treatment for more than 6 months. Next-generation sequencing identified homozygous deletions, deleterious mutations, or both in DNA-repair genes--including BRCA1/2, ATM, Fanconi's anemia genes, and CHEK2--in 16 of 49 patients who could be evaluated (33%). Of these 16 patients, 14 (88%) had a response to olaparib, including all 7 patients with BRCA2 loss (4 with biallelic somatic loss, and 3 with germline mutations) and 4 of 5 with ATM aberrations. The specificity of the biomarker suite was 94%. Anemia (in 10 of the 50 patients [20%]) and fatigue (in 6 [12%]) were the most common grade 3 or 4 adverse events, findings that are consistent with previous studies of olaparib. CONCLUSIONS: Treatment with the PARP inhibitor olaparib in patients whose prostate cancers were no longer responding to standard treatments and who had defects in DNA-repair genes led to a high response rate. (Funded by Cancer Research UK and others; ClinicalTrials.gov number, NCT01682772; Cancer Research UK number, CRUK/11/029.).