Amivantamab plus chemotherapy with and without lazertinib in EGFR-mutant advanced NSCLC after disease progression on osimertinib: primary results from the phase III MARIPOSA-2 study.

BACKGROUND: Amivantamab plus carboplatin-pemetrexed (chemotherapy) with and without lazertinib demonstrated antitumor activity in patients with refractory epidermal growth factor receptor (EGFR)-mutated advanced non-small-cell lung cancer (NSCLC) in phase I studies. These combinations were evaluated in a global phase III trial. PATIENTS AND METHODS: A total of 657 patients with EGFR-mutated (exon 19 deletions or L858R) locally advanced or metastatic NSCLC after disease progression on osimertinib were randomized 2 : 2 : 1 to receive amivantamab-lazertinib-chemotherapy, chemotherapy, or amivantamab-chemotherapy. The dual primary endpoints were progression-free survival (PFS) of amivantamab-chemotherapy and amivantamab-lazertinib-chemotherapy versus chemotherapy. During the study, hematologic toxicities observed in the amivantamab-lazertinib-chemotherapy arm necessitated a regimen change to start lazertinib after carboplatin completion. RESULTS: All baseline characteristics were well balanced across the three arms, including by history of brain metastases and prior brain radiation. PFS was significantly longer for amivantamab-chemotherapy and amivantamab-lazertinib-chemotherapy versus chemotherapy [hazard ratio (HR) for disease progression or death 0.48 and 0.44, respectively; P < 0.001 for both; median of 6.3 and 8.3 versus 4.2 months, respectively]. Consistent PFS results were seen by investigator assessment (HR for disease progression or death 0.41 and 0.38 for amivantamab-chemotherapy and amivantamab-lazertinib-chemotherapy, respectively; P < 0.001 for both; median of 8.2 and 8.3 versus 4.2 months, respectively). Objective response rate was significantly higher for amivantamab-chemotherapy and amivantamab-lazertinib-chemotherapy versus chemotherapy (64% and 63% versus 36%, respectively; P < 0.001 for both). Median intracranial PFS was 12.5 and 12.8 versus 8.3 months for amivantamab-chemotherapy and amivantamab-lazertinib-chemotherapy versus chemotherapy (HR for intracranial disease progression or death 0.55 and 0.58, respectively). Predominant adverse events (AEs) in the amivantamab-containing regimens were hematologic, EGFR-, and MET-related toxicities. Amivantamab-chemotherapy had lower rates of hematologic AEs than amivantamab-lazertinib-chemotherapy. CONCLUSIONS: Amivantamab-chemotherapy and amivantamab-lazertinib-chemotherapy improved PFS and intracranial PFS versus chemotherapy in a population with limited options after disease progression on osimertinib. Longer follow-up is needed for the modified amivantamab-lazertinib-chemotherapy regimen.

Associations of tissue tumor mutational burden and mutational status with clinical outcomes in KEYNOTE-042: pembrolizumab versus chemotherapy for advanced PD-L1-positive NSCLC.

BACKGROUND: We evaluated whether tissue tumor mutational burden (tTMB) and STK11, KEAP1, and KRAS mutations have clinical utility as biomarkers for pembrolizumab monotherapy versus platinum-based chemotherapy in patients with programmed death ligand 1 (PD-L1)-positive (tumor proportion score ≥1%) advanced/metastatic non-small-cell lung cancer (NSCLC) without EGFR/ALK alterations in the phase III KEYNOTE-042 trial. PATIENTS AND METHODS: This retrospective exploratory analysis assessed prevalence of tTMB and STK11, KEAP1, and KRAS mutations determined by whole-exome sequencing of tumor tissue and matched normal DNA and their associations with outcomes in KEYNOTE-042. Clinical utility of tTMB was assessed using a prespecified cut point of 175 mutations/exome. RESULTS: Of 793 patients, 345 (43.5%) had tTMB ≥175 mutations/exome and 448 (56.5%) had tTMB <175 mutations/exome. No association was observed between PD-L1 expression and tTMB. Continuous tTMB score was associated with improved overall survival (OS) and progression-free survival among patients receiving pembrolizumab (Wald test, one-sided P < 0.001) but not those receiving chemotherapy (Wald test, two-sided P > 0.05). tTMB ≥175 mutations/exome was associated with improved outcomes for pembrolizumab versus chemotherapy, whereas tTMB <175 mutations/exome was not {OS: hazard ratio, 0.62 [95% confidence interval (CI) 0.48-0.80] and 1.09 (95% CI 0.88-1.36); progression-free survival: 0.75 (0.59-0.95) and 1.27 (1.04-1.55), respectively}. Improved OS [hazard ratio (95% CI)] for pembrolizumab versus chemotherapy was observed regardless of STK11 [STK11 mutant (n = 33): 0.37 (0.16-0.86), STK11 wild-type (n = 396): 0.83 (0.65-1.05)]; KEAP1 [KEAP1 mutant (n = 64): 0.75 (0.42-1.35), KEAP1 wild-type (n = 365): 0.78 (0.61-0.99)], or KRAS [KRAS mutant (n = 69): 0.42 (0.22-0.81); KRAS wild-type (n = 232): 0.86 (0.63-1.18)] mutation status. CONCLUSION: tTMB with a cut point of ≥175 mutations/exome is a potential predictive biomarker for pembrolizumab monotherapy for advanced/metastatic PD-L1 tumor proportion score ≥1% NSCLC. Pembrolizumab is a standard first-line treatment in this setting regardless of STK11, KEAP1, or KRAS mutation status.

ARCTIC: durvalumab with or without tremelimumab as third-line or later treatment of metastatic non-small-cell lung cancer.

BACKGROUND: Many patients with metastatic non-small-cell lung cancer (mNSCLC) experience disease progression after first- and second-line treatment; more treatment options are required for these patients. ARCTIC, a phase III, randomized, open-label study, assessed durvalumab ± tremelimumab versus standard of care (SoC) as ≥ third-line treatment of mNSCLC. PATIENTS AND METHODS: ARCTIC comprised two independent sub-studies. Study A: 126 patients with ≥25% of tumor cells (TCs) expressing programmed cell death ligand-1 (PD-L1) were randomized (1 : 1) to durvalumab [up to 12 months 10 mg/kg every 2 weeks (q2w)] or SoC. Study B: 469 patients with PD-L1 TC <25% were randomized (3 : 2 : 2 : 1) to durvalumab + tremelimumab (12 weeks durvalumab 20 mg/kg + tremelimumab 1 mg/kg q4w then 34 weeks durvalumab 10 mg/kg q2w), SoC, durvalumab (up to 12 months 10 mg/kg q2w), or tremelimumab (24 weeks 10 mg/kg q4w then 24 weeks q12w). Primary end points: overall survival (OS) and progression-free survival (PFS) for durvalumab versus SoC (study A; descriptive only) and durvalumab + tremelimumab versus SoC (study B). RESULTS: Study A: median OS 11.7 (durvalumab) versus 6.8 (SoC) months {hazard ratio (HR) 0.63 [95% confidence interval (CI), 0.42-0.93]}; median PFS 3.8 (durvalumab) versus 2.2 (SoC) months [HR 0.71 (95% CI, 0.49-1.04)]. Study B: median OS 11.5 (durvalumab + tremelimumab) versus 8.7 (SoC) months [HR 0.80 (95% CI, 0.61-1.05); P = 0.109]. Median PFS of 3.5 months for both groups [HR 0.77 (95% CI, 0.59-1.01); P = 0.056]. Treatment-related grade 3/4 adverse events: 9.7% (durvalumab) and 44.4% (SoC; study A) and 22.0% (durvalumab + tremelimumab) and 36.4% (SoC; study B). CONCLUSIONS: In heavily pretreated patients with mNSCLC, durvalumab demonstrated clinically meaningful improvements in OS and PFS versus SoC (patients with PD-L1 TC ≥25%); numerical improvements in OS and PFS for durvalumab + tremelimumab versus SoC were observed (patients with PD-L1 TC <25%). Safety profiles were consistent with previous studies. TRIAL REGISTRATION: Clinicaltrials.gov identifier: NCT02352948.

Phase III safety study of intravenous NEPA: a novel fixed antiemetic combination of fosnetupitant and palonosetron in patients receiving highly emetogenic chemotherapy.

Background: NEPA, an oral fixed combination of the NK1RA netupitant (300 mg) and clinically/pharmacologically distinct 5-HT3RA palonosetron (PALO, 0.50 mg), is the first fixed antiemetic combination to have been approved. A single oral NEPA capsule plus dexamethasone (DEX) given before anthracycline-cyclophosphamide (AC) and non-AC highly emetogenic chemotherapy (HEC) showed superior prevention of chemotherapy-induced nausea and vomiting (CINV) over PALO plus DEX for 5 days postchemotherapy. The safety of NEPA was well-established in the phase II/III clinical program in 1169 NEPA-treated patients. An intravenous (i.v.) formulation of the NEPA combination (fosnetupitant 235 mg plus PALO 0.25 mg) has been developed. Patients and methods: This randomized, multinational, double-blind, stratified (by sex and country) phase III study (NCT02517021) in chemotherapy-naïve patients with solid tumors assessed the safety of a single dose of i.v. NEPA infused over 30 min before initial and repeated cycles of HEC. Patients received either i.v. NEPA or oral NEPA, both with oral DEX on days 1-4. Safety was assessed primarily by treatment-emergent adverse events (AEs) and electrocardiograms. Results: A total of 404 patients completed 1312 cycles. The incidence and type of treatment-emergent AEs were similar for both treatment groups with the majority of AEs as mild/moderate in intensity. There was no increased incidence of AEs in subsequent cycles in either group. The incidence of treatment-related AEs was similar and relatively low in both groups (12.8% i.v. NEPA and 11.4% oral NEPA during the entire study), with constipation being the most common (6.4% i.v. NEPA, 6.0% oral NEPA). No serious treatment-related AEs occurred in either group. No infusion site or anaphylactic reactions related to i.v. NEPA occurred. No clinically relevant changes in QTc and no cardiac safety concerns were observed. Conclusions: Intravenous NEPA was well-tolerated with a similar safety profile to oral NEPA in patients with various solid tumors receiving HEC.

A phase 1, randomized ascending single-dose study of antagonist anti-human CD40 ASKP1240 in healthy subjects.

This first-in-human, phase I study evaluated the safety, tolerability, pharmacokinetic and pharmacodynamic profile of ASKP1240 in healthy subjects. Twelve sequential groups (each 6 active and 3 placebo) were randomly assigned to placebo or single ascending doses of intravenous ASKP1240 (0.00003-10 mg/kg). ASKP1240 exhibited nonlinear pharmacokinetics, with mean maximal serum concentrations and area under the serum concentration-time curves ranging from 0.7 to 251.6 µg/mL and 6.5 to 55409.6 h·µg/mL following doses 0.1 mg/kg-10 mg/kg, respectively. CD40 receptor occupancy by ASKP1240, which was dose-dependent, reached a maximum at doses above 0.01 mg/kg. ASKP1240 was well tolerated, with no evidence of cytokine release syndrome or thromboembolic events. Treatment emergent antibodies to ASKP1240 were detected in 5/70 (7.1%) ASKP1240 recipients. In conclusion, antagonism of the CD40/CD154 interaction with ASKP1240 was safe and well tolerated at the doses tested.

Prediction of pouch of Douglas obliteration in women with suspected endometriosis using a new real-time dynamic transvaginal ultrasound technique: the sliding sign.

OBJECTIVE: To evaluate preoperative real-time dynamic transvaginal sonography (TVS) in the prediction of pouch of Douglas (POD) obliteration in women undergoing laparoscopy for suspected endometriosis. METHODS: This was a multicenter prospective observational study undertaken from January 2009 to November 2011. All women with symptoms suggestive of endometriosis who were scheduled for laparoscopy underwent detailed preoperative TVS, in particular to ascertain whether the POD was obliterated. POD obliteration was assessed using a real-time TVS technique called the 'sliding sign'. Preoperative TVS sliding sign findings were then compared to gold standard laparoscopic POD findings. RESULTS: One hundred consecutive women with preoperative TVS and laparoscopic outcomes were included in the final analysis. Mean age was 32.8 years and mean age at diagnosis of endometriosis was 27.4 years. At laparoscopy, 84/100 (84%) were found to have some form of endometriosis (73% peritoneal endometriosis, 35% ovarian endometrioma(s), 33% deep infiltrating endometriosis). At laparoscopy, 30/100 (30%) had an obliterated POD and 19/30 (63.3%) of these women also had evidence of bowel endometriosis. The sonographic sliding sign technique had an accuracy of 93.0%, sensitivity of 83.3%, specificity of 97.1%, positive predictive value of 92.6%, negative predictive value of 93.2%, positive likelihood ratio of 29.2 and negative likelihood ratio of 0.17 in the prediction of POD obliteration (P = 1.8E-16). CONCLUSIONS: Preoperative real-time dynamic TVS evaluation using the sliding sign seems to establish with a high degree of certainty whether the POD is obliterated. Given the increased risk of deep infiltrating endometriosis in women with POD obliteration, the TVS sliding sign technique may also be useful in the identification of women who may be at a higher risk for bowel endometriosis.

Metronomic oral vinorelbine in previously untreated advanced non-small-cell lung cancer patients unfit for platinum-based chemotherapy: results of the randomized phase II Tempo Lung trial.

BACKGROUND: To assess the efficacy and safety of a metronomic schedule of oral vinorelbine (mVNR) in advanced non-small-cell lung cancer (NSCLC) in patients unfit for platinum-based combination chemotherapy. PATIENTS AND METHODS: This was a multicenter, prospective, randomized, open-label phase II study in treatment-naive patients with TNM stage IIIB/IV NSCLC. Patients received mVNR at a fixed dose of 50 mg × 3 or standard schedule 60-80 mg/m2 weekly until disease progression or unacceptable toxicity. The primary endpoint was progression-free survival (PFS) without grade 4 toxicity (G4PFS; NCI-CTC v4). Main secondary objectives were safety, disease control rate (DCR) without grade 4 toxicity (G4DCR), DCR, PFS, overall survival (OS) and quality of life (QoL). RESULTS: A total of 167 patients were included, 83 and 84 patients in the mVNR and standard arms, respectively. The median G4PFS was 4.0 months [95% confidence interval (CI): 2.6-4.3] and 2.2 months (95% CI: 1.5-2.9), hazard ration (HR) = 0.63 (95% CI: 0.45-0.88), P = 0.0068 in favor of metronomic arm; G4DCR was 45.8% and 26.8% in the mVNR and standard arms, respectively. Grade 3-4 treatment-related adverse events were less frequent in the mVNR arm (25.3% versus 54.4%) mainly owing to a reduction in all grades (15.7% versus 51.9%) and grade 3-4 neutropenia (10.8% versus 42%). PFS was 4.3 (95% CI: 3.3-5.1) and 3.9 months (95% CI: 2.8-5.2) in mVNR and standard arms, respectively. No difference in median OS was observed. QoL was comparable between arms. CONCLUSIONS: Metronomic oral vinorelbine significantly prolonged median G4PFS in advanced NSCLC patients unfit for platinum combinations as first-line treatment. It was associated with a clear reduction in toxicity and may be considered as an important option in this challenging population.

Rationale and design of the multicentric, double-blind, double-placebo, randomized trial APrepitant versus HYdroxyzine in association with cytoreductive treatments for patients with myeloproliferative neoplasia suffering from Persistent Aquagenic Pruritus. Trial acronym: APHYPAP.

BACKGROUND: Aquagenic pruritus (AP), an intense sensation of scratching induced after water contact, is the most troublesome aspect of BCR-ABL1-negative myeloproliferative neoplasms (MPNs). Mostly described in polycythemia vera (PV, ~ 40%), it is also present in essential thrombocythemia (ET) and primary myelofibrosis (PMF) (10%). Even if this symptom can decrease or disappear under cytoreductive treatments, 30% of treated MPN patients still persist with a real impact on the quality of life (QoL). Because its pathophysiology is poorly understood, efficient symptomatic treatments of AP are missing. The neuropeptide substance P (SP) plays a crucial role in the induction of pruritus. Several studies showed the efficacy of aprepitant, an antagonist of SP receptor (NK-1R), in the treatment of chronic pruritus but never evaluated in AP. The objectives of APHYPAP are twofold: a clinical aim with the evaluation of the efficacy of two drugs in the treatment of a persistent AP for MPN patients and a biological aim to find clues to elucidate AP pathophysiology. METHODS/DESIGN: A multicentric, double-blind, double-placebo, randomized study will include 80 patients with MPN (PV or ET or PMF) treated since at least 6 months for their hemopathy but suffering from a persistent AP (VAS intensity ≥6/10). Patients will be randomized between aprepitant (80 mg daily) + placebo to match to hydroxyzine OR hydroxyzine (25 mg daily) + placebo to match to aprepitant for 14 days. At D0, baseline information will be collected and drugs dispense. Outcome measures will be assessed at D15, D30, D45, and D60. The primary study endpoint will be the reduction of pruritus intensity below (or equal) at 3/10 on VAS at D15. Secondary outcome measures will include the number of patients with a reduction or cessation of AP at D15 or D60; evaluation of QoL and AP characteristics at D0, D15, D30, D45, and D60 with MPN-SAF and AP questionnaires, respectively; modification of plasmatic concentrations of cytokines and neuropeptides at D0, D15, D30, and D60; and modification of epidermal innervation density and pruriceptor expression at D0 and D15. DISCUSSION: The APHYPAP trial will examine the efficacy of aprepitant vs hydroxyzine (reference treatment for AP) to treat persistent AP in MPN patients. The primary objective is to demonstrate the superiority of aprepitant vs hydroxyzine to treat persistent AP of MPN patients. The treatment received will be considered efficient if the AP intensity will be reduced at 3/10 or below on VAS after 14 days of treatment. The results of this study may provide a new treatment option for this troublesome symptom and also give us more insights in the pathophysiology understanding of AP. TRIAL REGISTRATION: APHYPAP. NCT03808805 , first posted: January 18, 2019; last update posted: June 10, 2021. EudraCT 2018-090426-66.

Fenfluramine-like cardiovascular side-effects of benfluorex.

Since 1976, benfluorex has been approved in Europe as a hypolipidemic and hypoglycemic drug, and is commonly used in the treatment of the metabolic syndrome. As a derivative of fenfluramine with an appetite suppressant action, benfluorex is preferentially used in overweight patients. In contrast to fenfluramine and dexfenfluramine, to date, benfluorex has not been reported to be associated with frequent cardiovascular side-effects. The present study reports five cases of severe pulmonary arterial hypertension and one case of valvular heart disease occurring in patients exposed to benfluorex. These individuals were middle age, diabetic females with a body mass index ranging 24.2-49 kg x m(-2). No definite causal effect for cardiovascular disease with benfluorex can be drawn from such case reports. However, as benfluorex, like dexfenfluramine and fenfluramine, is metabolised into active metabolite norfenfluramine, further extensive assessment of drug exposure in newly diagnosed pulmonary arterial hypertension or valvular heart disease patients is warranted.

Molecular beam scattering from liquid surfaces.

By means of controlled collisions of atoms and molecules with liquid surfaces, molecular beam experiments can be used to probe how gases stick to, rebound from, and exchange energy with molecules in the liquid phase. This report describes measurements of energy exchange in collisions between gases (neon, xenon, and sulfur hexafluoride) and polyatomic liquids (squalane and perfluoropolyether). Energy transfer depends critically on liquid composition and is more efficient for the hydrocarbon than for the perfluorinated ether.

Diagnostic value of plasma expression of microRNAs complementary to Drosha and Dicer in lung cancer patients.

OBJECTIVE: Lung cancer (LC) is diagnosed mostly in advanced, non-operable stage, with poor prognosis. The analysis of microRNAs may be a useful tool for early and non-invasive detection of cancer. Dicer and Drosha are enzymes with an essential role for microRNA biogenesis. The aim of our study was to analyze the expression of miRNA-27a-3p, miRNA-31, miRNA-182, miRNA-195 with the ability to reciprocal regulation of Dicer and Drosha expression in lung cancer patients. PATIENTS AND METHODS: The relative expression of microRNAs was detected by qPCR in plasma of 160 LC patients. The U-Mann Whitney test was used to compare the relative expression between particular groups of lung cancer patients and healthy individuals. The diagnostic value of microRNAs examination was analyzed using a receiver operating curve. RESULTS: We demonstrated that the plasma levels of miRNA-27, miRNA-31 and miRNA-182 were significantly higher and miRNA-195 significantly lower in the whole group of LC patients and in patients with early stages of NSCLC, in comparison with healthy donors. ROC analysis showed that four studied microRNAs have a potential diagnostic value for early stages of NSCLC with AUC=0.95 for miRNA-27a (94% sensitivity and 81% specificity, p=0.0001), 0.71 for miRNA-31 (73% sensitivity and 61% specificity, p=0.001) 0.77 for miRNA-182 (70% sensitivity and 79% specificity, p=0.0001) and 0.82 for miRNA-195 (74% sensitivity and 80% specificity, p=0.0001). CONCLUSIONS: We have proved that the expression of miRNA-27a-3p, miRNA-31, miRNA-182, and miRNA-195 in patients with LC is different from the expression of these molecules in healthy people. The examination of these microRNAs in plasma could be used in non-invasive lung cancer diagnosis.

Human tissue kallikrein 7, a novel biomarker for advanced ovarian carcinoma using a novel in situ quantitative method of protein expression.

BACKGROUND: Kallikreins, a subgroup of the serine protease enzyme family, are considered important prognostic biomarkers in cancer. Here, we sought to determine the prognostic value of kallikrein 7 (hk7) in ovarian cancer using a novel method of compartmentalized in situ protein analysis. PATIENTS AND METHODS: A tissue array composed of 150 advanced-stage ovarian cancers, uniformly treated with surgical debulking followed by platinum-paclitaxel (Taxol) combination chemotherapy, was constructed. For evaluation of kallikrein 7 protein expression, we used an immunofluorescence-based method of automated in situ quantitative measurement of protein analysis (AQUA). RESULTS: Mean follow-up time of the cohort was 34.35 months. One hundred and twenty eight of 150 cases had sufficient tissue for AQUA. In univariate survival analysis, low tumor hk7 expression was associated with better outcome for overall survival (OS) and disease-free survival in 3 years (P values 0.032 and 0.037, respectively). In multivariate survival analysis, adjusting for well-characterized prognostic variables, low tumor hk7 expression level was the most significant predictor variable for OS (95% confidence interval 0.125-0.729, P = 0.007). CONCLUSIONS: High tumor hk7 protein expression is associated with inferior patient outcome in ovarian cancer. hk7 may represent a promising prognostic factor in ovarian cancer.

Functional equivalency and diversity of cis-acting elements among yeast replication origins.

The DNA replication origins of the yeast Saccharomyces cerevisiae require several short functional elements, most of which are not conserved in sequence. To better characterize ARS305, a replicator from a chromosomal origin, we swapped functional DNA elements of ARS305 with defined elements of ARS1. ARS305 contains elements that are functionally exchangeable with ARS1 A and B1 elements, which are known to bind the origin recognition complex; however, the ARS1 A element differs in that it does not require a 3' box adjacent to the essential autonomously replicating sequence consensus. At the position corresponding to ARS1 B3, ARS305 has a novel element, B4, that can functionally substitute for every type of short element (B1, B2, and B3) in the B domain. Unexpectedly, the replacement of element B4 by ARS1 B3, which binds ABF1p and is known as a replication enhancer, inhibited ARS305 function. ARS305 has no short functional element at or near positions corresponding to the B2 elements in ARS1 and ARS307 but contains an easily unwound region whose functional importance was supported by a broad G+C-rich substitution mutation. Surprisingly, the easily unwound region can functionally substitute for the ARS1 B2 element, even though ARS1 B2 was found to possess a distinct DNA sequence requirement. The functionally conserved B2 element in ARS307 contains a known sequence requirement, and helical stability analysis of linker and minilinker mutations suggested that B2 also contains a DNA unwinding element (DUE). Our findings suggest that yeast replication origins employ a B2 element or a DUE to mediate a common function, DNA unwinding during initiation, although not necessarily through a common mechanism.

cis-acting components in the replication origin from ribosomal DNA of Saccharomyces cerevisiae.

The ribosomal DNA (rDNA) repeats of Saccharomyces cerevisiae contain an autonomously replicating sequence (ARS) that colocalizes with a chromosomal origin of replication. We show that a minimal sequence necessary for full ARS function corresponds to a 107-bp rDNA fragment which contains three 10-of-11-bp matches to the ARS consensus sequence. Point mutations in only one of the 10-of-11-bp matches, GTTTAT GTTTT, inactivate the rDNA ARS, indicating that this consensus sequence is essential. A perfect match to a revised ARS consensus is present but not essential. Sequences up to 9 bp 5' from the essential consensus are dispensable. A broad DNA region directly 3' to the essential consensus is required and is easily unwound as indicated by: (i) hypersensitivity to nicking of an approximately 100-bp region by mung bean nuclease in a negatively supercoiled plasmid and (ii) helical instability determined by thermodynamic analysis of the nucleotide sequence. A correlation between DNA helical instability and replication efficiency of wild-type and mutated ribosomal ARS derivatives suggests that a broad region 3' to the essential ARS consensus functions as a DNA unwinding element. Certain point mutations that do not stabilize the DNA helix in the 3' region but reduce ARS efficiency reveal an element distinct from, but overlapping, the DNA unwinding element. The nucleotide sequence of the functionally important constituents in the ARS appears to be conserved among the rDNA repeats in the chromosome.

DNA replication forks pause at silent origins near the HML locus in budding yeast.

Chromosomal replicators in budding yeast contain an autonomously replicating sequence (ARS) that functions in a plasmid, but certain ARSs are silent as replication origins in their natural chromosomal context. In chromosome III, the HML ARS cluster (ARS302-ARS303-ARS320) and ARS301 flank the transcriptionally silent mating-type locus HML, and all of these ARSs are silent as replication origins. ARS301 and ARS302 function in transcriptional silencing mediated by the origin recognition complex (ORC) and a heterochromatin structure, while the functions of ARS303 and ARS320 are not known. In this work, we discovered replication fork pause sites at the HML ARS cluster and ARS301 by analyzing DNA replication intermediates from the chromosome via two-dimensional gel electrophoresis. The replication fork pause at the HML ARS cluster was independent of cis- and trans-acting mutations that abrogate transcriptional silencing at HML. Deletion of the HML ARS cluster led to loss of the pause site. Insertion of a single, heterologous ARS (ARS305) in place of the HML ARS cluster reconstituted the pause site, as did multiple copies of DNA elements (A and B1) that bind ORC. The orc2-1 mutation, known to alter replication timing at origins, did not detectably affect the pause but activated the silent origin at the HML ARS cluster in a minority of cells. Delaying the time of fork arrival at HML led to the elimination of the pause sites at the HML ARS cluster and at the copy of ARS305 inserted in place of the cluster. Loss of the pause sites was accompanied by activation of the silent origins in the majority of cells. Thus, replication fork movement near HML pauses at a silent origin which is competent for replication initiation but kept silent through Orc2p, a component of the replication initiator. Possible functions for replication fork pause sites in checkpoints, S-phase regulation, mating-type switching, and transcriptionally silent heterochromatin are discussed.

Activation of silent replication origins at autonomously replicating sequence elements near the HML locus in budding yeast.

In the budding yeast, Saccharomyces cerevisiae, replicators can function outside the chromosome as autonomously replicating sequence (ARS) elements; however, within chromosome III, certain ARSs near the transcriptionally silent HML locus show no replication origin activity. Two of these ARSs comprise the transcriptional silencers E (ARS301) and I (ARS302). Another, ARS303, resides between HML and the CHA1 gene, and its function is not known. Here we further localized and characterized ARS303 and in the process discovered a new ARS, ARS320. Both ARS303 and ARS320 are competent as chromosomal replication origins since origin activity was seen when they were inserted at a different position in chromosome III. However, at their native locations, where the two ARSs are in a cluster with ARS302, the I silencer, no replication origin activity was detected regardless of yeast mating type, special growth conditions that induce the transcriptionally repressed CHA1 gene, trans-acting mutations that abrogate transcriptional silencing at HML (sir3, orc5), or cis-acting mutations that delete the E and I silencers containing ARS elements. These results suggest that, for the HML ARS cluster (ARS303, ARS320, and ARS302), inactivity of origins is independent of local transcriptional silencing, even though origins and silencers share key cis- and trans-acting components. Surprisingly, deletion of active replication origins located 25 kb (ORI305) and 59 kb (ORI306) away led to detection of replication origin function at the HML ARS cluster, as well as at ARS301, the E silencer. Thus, replication origin silencing at HML ARSs is mediated by active replication origins residing at long distances from HML in the chromosome. The distal active origins are known to fire early in S phase, and we propose that their inactivation delays replication fork arrival at HML, providing additional time for HML ARSs to fire as origins.

QT Interval Shortening With Isavuconazole: In Vitro and In Vivo Effects on Cardiac Repolarization.

The effects of isavuconazole (active moiety of isavuconazonium sulfate) on cardiac ion channels in vitro and cardiac repolarization clinically were assessed in a phase I, randomized, double-blind study in healthy individuals who received isavuconazole (after 2-day loading dose), at therapeutic or supratherapeutic doses daily for 11 days, moxifloxacin (400 mg q.d.), or placebo. A post-hoc analysis of the phase III SECURE trial assessed effects on cardiac safety. L-type Ca2+ channels were most sensitive to inhibition by isavuconazole. The 50% inhibitory concentrations for ion channels were higher than maximum serum concentrations of nonprotein-bound isavuconazole in vivo. In the phase I study (n = 161), isavuconazole shortened the QT interval in a dose- and plasma concentration-related manner. There were no serious treatment-emergent adverse events; palpitations and tachycardia were observed in placebo and supratherapeutic isavuconazole groups; no cardiac safety signals were detected in the SECURE study (n = 257). Isavuconazole was associated with a shortened cardiac QT interval.

The inefficient replication origin from yeast ribosomal DNA is naturally impaired in the ARS consensus sequence and in DNA unwinding.

Ribosomal DNA (rDNA) replication origins of Saccharomyces cerevisiae are known to function inefficiently, both in the context of the tandem rDNA repeats in the chromosome and as single copy autonomously replicating sequences (ARSs) in plasmids. Here we examined components of the rDNA ARS that might contribute to inefficient extrachromosomal replication. Like the efficient H4 ARS, the rDNA ARS requires a match to the 11 bp ARS consensus sequence (ACS) and a broad non-conserved region that may contain multiple elements, including a DNA unwinding element (DUE). Using a single-strand-specific nuclease hypersensitivity assay and by determining the superhelical density required for stable DNA unwinding, we found that the DNA of the rDNA ARS is not as easily unwound as the H4 ARS. Unwinding of the rDNA ARS required additional energy, similar to the unwinding of mutations in the H4 ARS that stabilize the double helix in the DUE region and impair replication. In vivo extrachromosomal replication of the rDNA ARS was cold sensitive, like H4 ARS mutants that require additional energy to unwind the DUE region but unlike the easily unwound, wild-type H4 ARS. Impairment of replication function at reduced temperature suggests that the elevated energy requirement for DNA unwinding inherent in the wild-type rDNA ARS contributes to inefficient replication function. We also examined the essential ACS match in the rDNA ARS, which is known to be imperfect at one position. A point mutation in the essential ACS that corrects the imperfect match increased the efficiency of extrachromosomal replication. Our results reveal that the essential ACS element and DNA unwinding in the rDNA ARS are naturally impaired, suggesting that inefficient function of the rDNA replication origin has a biological purpose.

Antitumor drug adozelesin differentially affects active and silent origins of DNA replication in yeast checkpoint kinase mutants.

The antitumor drug adozelesin is a potent cytotoxic DNA-damaging agent. Here we determined how adozelesin affects chromosomal DNA replication at a molecular level in a yeast model system and examined the influence of checkpoint kinase genes, the human homologues of which are mutated in cancer. Analysis of replication intermediates using two-dimensional gel electrophoresis showed that adozelesin inhibited the activity of a replication origin and stalled replication fork progression through chromosomal DNA at the origin. RAD53 and MEC1 protein kinase genes, homologues of human CHK2 and ATM, respectively, regulate an intra-S-phase DNA damage checkpoint and, when mutated, permit unchecked replication of damaged DNA in S-phase. Mutations in these genes did not abrogate adozelesin-induced inhibition of origin activity and fork progression at the replication origin. However, novel replication intermediates indicative of DNA breaks were detected only in the rad53 mutant, suggesting a role for the wild-type gene in maintaining chromosome integrity in the presence of the drug. In contrast to the inhibition of the active replication origin by adozelesin, normally silent origins present in the same chromosome were activated by adozelesin in rad53 and mec1 mutant cells. Thus, an antitumor drug that damages DNA can induce an abnormal replication pattern in a chromosome by activating silent origins, depending upon defects in yeast checkpoint kinase genes, the homologues of which are mutated in cancer. Implications of an abnormal replication pattern for the epigenetic regulation of gene expression are discussed.

DNA helical instability facilitates initiation at the SV40 replication origin.

Previous analysis of mutations in bacterial and yeast replication origins has identified a genetic component, termed a DNA unwinding element (DUE), whose intrinsic helical instability is essential for origin function. For the SV40 replication origin, we show here that the early palindrome (EP) and A + T-rich (AT) domains both exhibit helical instability, despite their dissimilar A + T compositions. To test the possible contribution of helical instability to SV40 origin function, the relationship between helical stability of mutant origin sequences and their known origin activity in vitro and in vivo was examined. Origin activity correlates inversely with the helical stability of mutations within the EP domain but not the AT domain or the T-antigen binding domain. The quantitative correlation holds for four different measures of origin activity determined in vitro and in vivo. An even better-correlated collection of mutations was found in a specific portion of the EP domain. This specific EP subdomain coincides with the sequence known to be strand-separated after T-antigen binds the origin in vitro and with the origin of bidirectional replication in vivo. Our analysis of origin mutations indicates that the helical instability of the specific EP subdomain is required to facilitate T-antigen-induced melting and the initiation of DNA replication. The sensitivity of the required EP subdomain to mutations that stabilize the DNA helix defines the DUE of the SV40 replication origin.