A modular and synthetic biosynthesis platform for de novo production of diverse halogenated tryptophan-derived molecules.

Halogen-containing molecules are ubiquitous in modern society and present unique chemical possibilities. As a whole, de novo fermentation and synthetic pathway construction for these molecules remain relatively underexplored and could unlock molecules with exciting new applications in industries ranging from textiles to agrochemicals to pharmaceuticals. Here, we report a mix-and-match co-culture platform to de novo generate a large array of halogenated tryptophan derivatives in Escherichia coli from glucose. First, we engineer E. coli to produce between 300 and 700 mg/L of six different halogenated tryptophan precursors. Second, we harness the native promiscuity of multiple downstream enzymes to access unexplored regions of metabolism. Finally, through modular co-culture fermentations, we demonstrate a plug-and-play bioproduction platform, culminating in the generation of 26 distinct halogenated molecules produced de novo including precursors to prodrugs 4-chloro- and 4-bromo-kynurenine and new-to-nature halogenated beta carbolines.

Fluorescence-Based Screens for Engineering Enzymes Linked to Halogenated Tryptophan.

Directed evolution is often limited by the throughput of accurate screening methods. Here we demonstrate the feasibility of utilizing a singular transcription factor (TF)-system that can be refactored in two ways (both as an activator and repressor). Specifically, we showcase the use of previously evolved 5-halo- or 6-halo-tryptophan-specific TF biosensors suitable for the detection of a halogenated tryptophan molecule in vivo. We subsequently validate the biosensor's utility for two halogenase-specific halo-tryptophan accumulation screens. First, we isolated 5-tryptophan-halogenase, XsHal, from a mixed pool of halogenases with 100% efficiency. Thereafter, we generated a targeted library of the catalytic residue of 6-tryptophan halogenase, Th-Hal, and isolated functioning halogenases with 100% efficiency. Lastly, we refactor the TF circuit to respond to the depletion of halogenated tryptophan and prototype a high-throughput biosensor-directed evolution scheme to screen for downstream enzyme variants capable of promiscuously converting halogenated tryptophan. Altogether, this work takes a significant step toward the rapid and higher throughput screening of halogenases and halo-tryptophan converting enzymes to further reinforce efforts to enable high-level bioproduction of halogenated chemicals.

Abemaciclib in patients with p16ink4A-deficient mesothelioma (MiST2): a single-arm, open-label, phase 2 trial.

BACKGROUND: Genetically stratified therapy for malignant mesothelioma is unavailable. Mesotheliomas frequently harbour loss of the chromosome 9p21.3 locus (CDKN2A-MTAP), which is associated with shorter overall survival due to loss of the tumour suppressor p16ink4A, an endogenous suppressor of cyclin-dependent kinase (CDK)4 and CDK6. Genetic restoration of p16ink4A suppresses mesothelioma in preclinical models, underpinning the rationale for targeting CDK4 and CDK6 in p16ink4A-negative mesothelioma. We developed a multicentre, stratified, phase 2 trial to test this hypothesis. METHODS: The MiST2 study was a single-arm, open-label, phase 2 clinical trial done two UK centres. Patients older than 18 years with any histologically confirmed subtype of mesothelioma (pleural or peritoneal) with radiological progression after at least one course of platinum-based chemotherapy were molecularly screened by immunohistochemistry for p16ink4A. Patients with p16ink4A-negative mesothelioma were eligible for inclusion in the study. Patients were required to have measurable disease by modified Response Evaluation Criteria in Solid Tumours version 1.1 for malignant mesothelioma, a predicted life expectancy of at least 12 weeks, and an Eastern Cooperative Oncology Group performance status score of 0-1. Patients received oral abemaciclib 200 mg twice daily, administered in 28-day cycles for 24 weeks. The primary endpoint was the disease control rate (patients with complete responses, partial responses, or stable disease) at 12 weeks. The null hypothesis could be rejected if at least 11 patients had disease control. The efficacy and safety populations were defined as all patients who received at least one dose of the study drug. The study is registered with ClinicalTrials.gov, NCT03654833, and is ongoing (but MiST2 is now closed). FINDINGS: Between Sept 31, 2019, and March 2, 2020, 27 eligible patients consented to molecular screening. The median follow-up was 18·4 weeks (IQR 6·7-23·9). One patient was excluded before treatment because of a serious adverse event before study drug allocation. 26 (100%) of 26 treated patients were p16ink4A deficient and received at least one dose of abemaciclib. Disease control at 12 weeks was reported in 14 (54%) of 26 patients (95% CI 36-71). Grade 3 or worse treatment-related adverse events (of any cause) occurred in eight (27%) of 26 patients (diarrhoea, dyspnoea, thrombocytopenia, vomiting, urinary tract infection, increased alanine aminotransferase, ascites, chest infection or suspected chest infection, neutropenic sepsis, alopecia, blood clot left calf, fall [broken neck and collar bone], haemoptysis, lower respiratory tract infection, and pulmonary embolism). Grade 3 or worse treatment-related adverse events occurred in three (12%) of 26 patients (diarrhoea, thrombocytopenia, vomiting, increased alanine aminotransferase, and pulmonary embolism). Serious adverse events occurred in six (23%) of 26 patients, leading to treatment discontinuation in one (4%) patient (diarrhoea, urinary tract infection, chest infection, neutropenic sepsis, fall [broken neck and collar bone], haemoptysis, lower respiratory tract infection, and pulmonary embolism). One patient had a serious adverse event related to abemaciclib (diarrhoea). One (4%) of 26 patients died from an adverse event (neutropenic sepsis). INTERPRETATION: This study met its primary endpoint, showing promising clinical activity of abemaciclib in patients with p16ink4A-negative mesothelioma who were previously treated with chemotherapy, and warrants its further investigation in a randomised study as a targeted stratified therapy. FUNDING: University of Leicester, Asthma UK and British Lung Foundation Partnership, and the Victor Dahdaleh Foundation.

Nivolumab versus placebo in patients with relapsed malignant mesothelioma (CONFIRM): a multicentre, double-blind, randomised, phase 3 trial.

BACKGROUND: No phase 3 trial has yet shown improved survival for patients with pleural or peritoneal malignant mesothelioma who have progressed following platinum-based chemotherapy. The aim of this study was to assess the efficacy and safety of nivolumab, an anti-PD-1 antibody, in these patients. METHODS: This was a multicentre, placebo-controlled, double-blind, parallel group, randomised, phase 3 trial done in 24 hospitals in the UK. Adult patients (aged ≥18 years) with an Eastern Cooperative Oncology Group performance status of 0 or 1, with histologically confirmed pleural or peritoneal mesothelioma, who had received previous first-line platinum-based chemotherapy and had radiological evidence of disease progression, were randomly assigned (2:1) to receive nivolumab at a flat dose of 240 mg every 2 weeks over 30 min intravenously or placebo until disease progression or a maximum of 12 months. The randomisation sequence was generated within an interactive web response system (Alea); patients were stratified according to epithelioid versus non-epithelioid histology and were assigned in random block sizes of 3 and 6. Participants and treating clinicians were masked to group allocation. The co-primary endpoints were investigator-assessed progression-free survival and overall survival, analysed according to the treatment policy estimand (an equivalent of the intention-to-treat principle). All patients who were randomly assigned were included in the safety population, reported according to group allocation. This trial is registered with Clinicaltrials.gov, NCT03063450. FINDINGS: Between May 10, 2017, and March 30, 2020, 332 patients were recruited, of whom 221 (67%) were randomly assigned to the nivolumab group and 111 (33%) were assigned to the placebo group). Median follow-up was 11·6 months (IQR 7·2-16·8). Median progression-free survival was 3·0 months (95% CI 2·8-4·1) in the nivolumab group versus 1·8 months (1·4-2·6) in the placebo group (adjusted hazard ratio [HR] 0·67 [95% CI 0·53-0·85; p=0·0012). Median overall survival was 10·2 months (95% CI 8·5-12·1) in the nivolumab group versus 6·9 months (5·0-8·0) in the placebo group (adjusted HR 0·69 [95% CI 0·52-0·91]; p=0·0090). The most frequently reported grade 3 or worse treatment-related adverse events were diarrhoea (six [3%] of 221 in the nivolumab group vs two [2%] of 111 in the placebo group) and infusion-related reaction (six [3%] vs none). Serious adverse events occurred in 90 (41%) patients in the nivolumab group and 49 (44%) patients in the placebo group. There were no treatment-related deaths in either group. INTERPRETATION: Nivolumab represents a treatment that might be beneficial to patients with malignant mesothelioma who have progressed on first-line therapy. FUNDING: Stand up to Cancer-Cancer Research UK and Bristol Myers Squibb.

Clonal architecture in mesothelioma is prognostic and shapes the tumour microenvironment.

Malignant Pleural Mesothelioma (MPM) is typically diagnosed 20-50 years after exposure to asbestos and evolves along an unknown evolutionary trajectory. To elucidate this path, we conducted multi-regional exome sequencing of 90 tumour samples from 22 MPMs acquired at surgery. Here we show that exomic intratumour heterogeneity varies widely across the cohort. Phylogenetic tree topology ranges from linear to highly branched, reflecting a steep gradient of genomic instability. Using transfer learning, we detect repeated evolution, resolving 5 clusters that are prognostic, with temporally ordered clonal drivers. BAP1/-3p21 and FBXW7/-chr4 events are always early clonal. In contrast, NF2/-22q events, leading to Hippo pathway inactivation are predominantly late clonal, positively selected, and when subclonal, exhibit parallel evolution indicating an evolutionary constraint. Very late somatic alteration of NF2/22q occurred in one patient 12 years after surgery. Clonal architecture and evolutionary clusters dictate MPM inflammation and immune evasion. These results reveal potentially drugable evolutionary bottlenecking in MPM, and an impact of clonal architecture on shaping the immune landscape, with potential to dictate the clinical response to immune checkpoint inhibition.

Rucaparib in patients with BAP1-deficient or BRCA1-deficient mesothelioma (MiST1): an open-label, single-arm, phase 2a clinical trial.

BACKGROUND: Malignant mesothelioma remains an incurable cancer, with no effective treatments in the setting of relapsed disease. Homologous recombination deficiency predicts sensitivity to poly (ADP-ribose) polymerase (PARP) inhibitors. In mesothelioma, BRCA1-associated protein 1 carboxy-terminal hydrolase (BAP1), which regulates DNA repair, is frequently mutated. We aimed to test the hypothesis that BAP1-deficient or BRCA1-deficient mesotheliomas would be sensitive to PARP inhibition by rucaparib. METHODS: We did a single-centre, open-label, single-arm, phase 2a trial in Leicester, UK, with prospective molecular stratification (Mesothelioma-Stratified Therapy 1 [MiST1]). Patients aged 18 years or older who had radiologically progressing, histologically confirmed, malignant mesothelioma after at least one course of systemic treatment; with cytoplasmic-BAP1-deficient or BRCA1-deficient mesothelioma (pleural or peritoneal or other primary localisation), and who met the other inclusion criteria, were deemed eligible. All eligible patients who consented to take part were given rucaparib 600 mg twice a day orally, for six cycles of 28 days, or until disease progression, unacceptable toxicity, withdrawal of consent, or death. Response was measured by CT scan every 6 weeks. The primary outcome was disease control (complete response, partial response, or stable disease) at 12 weeks in all patients who received study drug; secondary outcomes were the safety and toxicity profile, objective response rate (proportion of complete or partial responses), and disease control rate at 24 weeks. Recruitment is now closed. This trial is registered with ClinicalTrials.gov, NCT03654833. FINDINGS: Between Feb 9 and June 10, 2019, we enrolled 26 molecularly and clinically eligible patients. Ten (38%) of 26 patients were BAP1 negative and BRCA1 negative, 23 patients (89%) were BAP1 negative, and 13 patients (50%) were BRCA1 negative. Disease control rate at 12 weeks was 58% (95% CI 37-77; 15 of 26 patients), and at 24 weeks was 23% (9-44; six of 26 patients). Rucaparib was well tolerated, with 15 (9%) of 166 adverse events being grade 3 or 4, which were seen in nine (35%) of 26 patients, and there were no deaths. The most common grade 1-2 adverse events were nausea (18 [69%] of 26 patients), fatigue (14 patients [54%]), and decreased appetite (ten patients [38%]). The most common grade 3-4 adverse events were upper respiratory tract infection (three patients [12%]) and anaemia (three patients [12%]). All six cycles of rucaparib were received by eight (31%) of 26 patients. One or more dose reductions occurred in nine patients (35%). INTERPRETATION: Rucaparib in patients with BAP1-negative or BRCA1-negative mesothelioma met the prespecified criteria for success, showing promising activity with manageable toxicity. Further investigation of homologous recombination deficiency mutations is planned to refine the identification of predictive biomarkers for PARP inhibition in mesothelioma. FUNDING: University of Leicester (Leicester, UK), Asthma UK and British Lung Foundation Partnership, and the Victor Dahdaleh Foundation (Toronto, ON, Canada).

Women on Hormone Therapy with Ischemic Stroke, Effects on Deficits and Recovery.

BACKGROUND: Hormone replacement therapy (HT) for post-menopausal women is associated with increased incidence of ischemic stroke risk. Effects of HT on stroke related deficits and functional outcomes in acute ischemic stroke (AIS) are uncertain. We retrospectively examined female consult data for HT use and National Institutes of Health Stroke Score (NIHSS) at baseline and recovery for 2015 and 2016 in a large stroke telemedicine program. HYPOTHESIS: The age of women who acknowledged HT use will negatively impact stroke severity and outcomes. METHODS: We analyzed consult data from two consecutive years for all women and included HT use, current age, and baseline and 24 h NIHSS's. We included all women consults regardless of IV Alteplase treatment. 24 h NIHSS and three month modified Rankin scale (mRS) were included from women given IV Alteplase. RESULTS: Strokes were identified in 523 women and 244 women received Alteplase therapy. Women without HT use numbered 459 and 64 women listed HT use. Mean NIHSS scores regardless of HT use significantly improved 24 h NIHSS vs. baseline NIHSS (p<0.0001). Baseline NIHSS scores were significantly improved in women on HT vs. non-HT users (p=0.01) in women age 50 to 79 years. Although mean NIHSS scores at 24h was not different from HT to no HT use (4.9 ± 1.6 vs. 7.8 ± 0.6, p=0.08) a trend was present for lower NIHSS scores for women 50-79 years. The mRS scores at three months indicated significant improvements among HT users vs. non-HT use (1.46 ± 0.4 vs. 2.51 ± 0.2, p=0.05). CONCLUSION: While cautions persist on the use, route and dosage of HT for risks of ischemic stroke, the HT moderation of AIS deficits and outcomes in women <80 years of age warrants further investigation.

A small multimarker panel using simple immunohistochemistry methods is an adjunct to stage for cutaneous melanoma prognosis.

Melanoma is an aggressive cancer. Outcomes can vary significantly for lesions within the same pathological stage - a problem of increasing relevance with the promise of adjuvant treatments on the basis of immune checkpoint modulators and targeted therapies. The use of a panel of prognostic molecular biomarkers as an adjunct to stage represents a possible solution. Immunohistochemistry-based biomarkers offer greater potential for translation into clinical practice than biomarkers utilizing more complex methods. Many immunohistochemistry-based biomarkers have been identified through discovery studies, but rigorous validation of these is scarce. We take the first steps towards validating a combination of three such biomarkers in a prognostic panel - 5hmC, ki-67 and p16. Immunohistochemistry was performed on a cohort of 50 melanomas to determine the expression of 5hmC, ki-67 and p16. 5hmC and p16 showed statistically significant differences in metastasis-free survival between low-score and high-score groups, whereas the use of all three biomarkers together with stage could predict the 5-year metastasis risk more accurately than stage alone. Our results suggest that the use of multimarker panels to improve the accuracy of prognostic predictions is feasible and worthy of further study. We have shown that a small immunohistochemistry-based panel utilizing simple, inexpensive, reproducible methods can be an effective adjunct to stage in prognostic prediction. A follow-up study consisting of a large cohort of melanomas is now indicated to continue the development of the prognostic panel.

Alternative dosing of prophylactic enoxaparin in the trauma patient: is more the answer?

BACKGROUND: Inadequate anti-factor Xa levels and increased venous thromboembolic events occur in trauma patients receiving standard prophylactic enoxaparin dosing. The aim of this study was to test the hypothesis that higher dosing (40 mg twice daily) would improve peak anti-Xa levels and decrease venous thromboembolism. METHODS: A retrospective review was performed of trauma patients who received prophylactic enoxaparin and peak anti-Xa levels over 27 months. Patients were divided on the basis of dose: group A received 30 mg twice daily, and group B received 40 mg twice daily. Demographics and rates of venous thromboembolism were compared between dose groups and patients with inadequate or adequate anti-Xa levels. RESULTS: One hundred twenty-four patients were included, 90 in group A and 34 in group B. Demographics were similar, except that patients in group B had a higher mean body weight. Despite this, only 9% of group B patients had inadequate anti-Xa levels, compared with 33% of those in group A (P = .01). Imaging studies were available in 69 patients and revealed 8 venous thromboembolic events (P = NS, group A vs group B) with significantly more venous thromboembolic events occurring in patients with low anti-Xa levels (P = .02). CONCLUSIONS: Although higher dosing of enoxaparin led to improved anti-Xa levels, this did not equate to a statistical decrease in venous thromboembolism.