Altering lamina assembly reveals lamina-dependent and -independent functions for A-type lamins.

Lamins are intermediate filament proteins that form a fibrous meshwork, called the nuclear lamina, between the inner nuclear membrane and peripheral heterochromatin of metazoan cells. The assembly and incorporation of lamin A/C into the lamina, as well as their various functions, are still not well understood. Here, we employed designed ankyrin repeat proteins (DARPins) as new experimental tools for lamin research. We screened for DARPins that specifically bound to lamin A/C, and interfered with lamin assembly in vitro and with incorporation of lamin A/C into the native lamina in living cells. The selected DARPins inhibited lamin assembly and delocalized A-type lamins to the nucleoplasm without modifying lamin expression levels or the amino acid sequence. Using these lamin binders, we demonstrate the importance of proper integration of lamin A/C into the lamina for nuclear mechanical properties and nuclear envelope integrity. Finally, our study provides evidence for cell-type-specific differences in lamin functions.

Human caspases in vitro: expression, purification and kinetic characterization.

A number of strategies and protocols for the expression, purification and kinetic characterization of human caspases are described in the literature. We have systematically revised these protocols and present comprehensive optimized expression and purification protocols for caspase-1 to -9 as well as improved assay conditions for their reproducible kinetic characterization. Our studies on active site titration revealed that the reproducibility is strongly affected by the presence of DTT in the assay buffer. Furthermore, we observed that not all caspases show a linear relationship between enzymatic activity and protein concentration, which explains the discrepancy between published values of specific activities from different laboratories. Our broad kinetic analysis allows the conclusion that the dependency of caspase activities on protein concentration is an effect of concentration-dependent dimerization, which can also be influenced by kosmotropic salts. The protocol recommendations as an outcome of this work will yield higher reproducibility regarding expression and purification of human caspases and contribute to standardization of enzyme kinetic data.

A prognostic score for patients with malignant pleural mesothelioma (MPM) receiving second-line immunotherapy or chemotherapy in the ETOP 9-15 PROMISE-meso phase III trial.

INTRODUCTION: Clinical and laboratory parameters associated with response for patients with advanced pre-treated malignant pleural mesothelioma (MPM) are lacking. We aimed to identify prognostic and predictive markers among patients with relapsed MPM who were randomised into the ETOP 9-15 PROMISE-meso phase III trial, evaluating pembrolizumab and chemotherapy. METHODS: Baseline clinical and laboratory parameters were investigated for prognostic or predictive value on progression-free survival (PFS) and overall survival (OS) in a retrospective analysis, based on the full cohort of 144 MPM patients. These consisted of immune-inflammatory indexes (neutrophil-lymphocyte ratio [NLR], systemic immune-inflammatory index [SII], lactate dehydrogenase [LDH]) along with other already known prognostic baseline characteristics and laboratory values. Cut-offs were chosen independently of outcome. Based on Cox multivariable analysis for PFS in the whole cohort, a risk factor model was built to illustrate the prognostic stratification of patients by the combination of the derived independent prognostic factors, taking into account the EORTC score, a validated prognostic score in MPM. All models were stratified by histology and adjusted by treatment. RESULTS: In the stratified multivariable analysis in the whole cohort, high SII (hazard ratio (HR) 2.06; 95%CI 1.39-3.05) and low haemoglobin (HR 1.62; 95%CI 1.06-2.50) were associated with worse PFS. Based on these two prognostic factors, a mesothelioma risk score (MRS) was constructed with three PFS risk prognosis categories: favourable, intermediate and poor with 0, 1 and 2 risk factors, respectively (corresponding percent of cohort: 24%, 34% and 42% and median PFS: 5.8, 4.2 and 2.1 months). The derived MRS stratified the prognosis for PFS and OS, overall and within each of the EORTC groups. No significant predictors of treatment benefit were identified. CONCLUSIONS: The proposed MRS is prognostic of patient outcome and it fine-tunes the prognosis of patients with pre-treated MPM alone or when used with the already established EORTC score.

Alectinib for the treatment of pretreated RET-rearranged advanced NSCLC: Results of the ETOP ALERT-lung trial.

BACKGROUND: Alectinib, a highly selective next generation ALK-inhibitor, has exhibited potent anti-tumour activity in RET-rearranged NSCLC in the preclinical stage. METHODS: ALERT-lung is a single-arm, phase II trial evaluating the activity of alectinib for the treatment of pretreated RET-rearranged advanced NSCLC. Alectinib was administered orally, 600 mg, twice per day until progression, refusal or unacceptable toxicity (treatment could continue beyond progression, if patient was deriving clinical benefit). Patient recruitment closed prematurely due to discouraging results for alectinib in a phase I/II study in the same indication. RESULTS: All 14 patients who enrolled until the premature accrual closure, received at lease one dose of alectinib. Among them, median age was 61 years, majority (71 %) was female, never smokers, of ECOG PS 1. No objective response (complete or partial response) was recorded. Of the 13 evaluable patients, three (23 %) achieved and maintained disease stabilisation for 24 weeks. Up to 31 March 2021 (median follow-up 15.9 months), 12 PFS-events (92 %) were observed, with median PFS of 3.7 months (95 % C.I.: 1.8 - 7.3 months). Overall, three deaths (23 %) were reported. Seven patients (50 %) experienced grade ≥ 3 adverse events, while three discontinued treatment due to erythema multiforme of grade 3, related to alectinib. No treatment-related serious adverse event was reported. CONCLUSIONS: Accrual into our trial was terminated early in response to other reports of limited activity of alectinib in patients with RET-fusion NSCLC and the emergence of more potent selective RET-inhibitors. Also in our trial, alectinib did not show the expected potential for anti-tumour activity in NSCLC.

Combined, patient-level, analysis of two randomised trials evaluating the addition of denosumab to standard first-line chemotherapy in advanced NSCLC - The ETOP/EORTC SPLENDOUR and AMGEN-249 trials.

INTRODUCTION: The efficacy of adding denosumab to standard first-line chemotherapy for advanced NSCLC patients has been evaluated in two separate randomised trials (SPLENDOUR and AMGEN-249). In this pooled analysis, we will assess the combination-treatment effect in the largest available population, in order to conclude about the potential impact of denosumab in NSCLC. METHODS: Both trials included in this combined analysis, were randomised (SPLENDOUR 1:1, AMGEN-249 2:1) multi-centre trials stratified by histology, bone metastasis, geographical region and for SPLENDOUR only, ECOG PS. Cox proportional hazards models, were used to assess the treatment effect with respect to overall survival (OS; primary endpoint) and progression-free survival (PFS; secondary endpoint). Heterogeneity between trials was assessed, and subgroup analyses were performed. RESULTS: The pooled analysis was based on 740 randomised patients (SPLENDOUR:514; AMGEN-249:226), with 407 patients in the chemotherapy-denosumab arm and 333 in the chemotherapy-alone arm. In the chemotherapy-denosumab arm, at a median follow-up of 22.0 months, 277 (68.1%) deaths were reported with median OS 9.2 months (95%CI:[8.0-10.7]), while in the chemotherapy-alone arm, with similar median follow-up of 20.3 months, 230 (69.1%) deaths with median OS 9.9 months (95%CI:[8.2-11.2]). No significant denosumab effect was found (HR = 0.98; 95%CI:[0.82-1.18]; P = 0.85). Among subgroups, interaction was found between treatment and histology subtypes (P = 0.020), with a statistically significant benefit in the squamous group (HR = 0.70; 95%CI:[0.49-0.98]; P = 0.038), from 7.6 to 9.0 months median OS. With respect to PFS, 363 (89.2%) and 298 (89.5%) events were reported in the chemotherapy-denosumab and chemotherapy-alone arms, respectively, with corresponding medians 4.8 months (95%CI:[4.4-5.3]) and 4.9 months (95%CI:[4.3-5.4]). HR for PFS was 0.97(95%CI:[0.83-1.15]; P = 0.76), indicating that no significant denosumab benefit existed for PFS. CONCLUSION: In this pooled analysis, no statistically significant improvement was shown in PFS/OS with the combination of denosumab and chemotherapy for advanced NSCLC and no meaningful benefit in any of the subgroups.

Progression-Free and Overall Survival for Concurrent Nivolumab With Standard Concurrent Chemoradiotherapy in Locally Advanced Stage IIIA-B NSCLC: Results From the European Thoracic Oncology Platform NICOLAS Phase II Trial (European Thoracic Oncology Platform 6-14).

INTRODUCTION: The NICOLAS study is the first completed single-arm phase II trial in stage III NSCLC evaluating hierarchically first the safety and then the efficacy of adding nivolumab concurrently to standard definitive concurrent chemoradiotherapy. The safety end point was reported earlier; here, we present the efficacy results. METHODS: Stage IIIA-B unresectable treatment-naive patients with NSCLC received three cycles of platinum-based chemotherapy and concurrent radiotherapy (66 Gy, 33 fractions), along with nivolumab (360 mg, 3-weekly). Nivolumab was continued as monotherapy consolidation for a maximum of 1 year (480 mg, 4-weekly). The primary end point was 1-year progression-free survival (PFS), with a target improvement compared with historical data of at least 15%, from 45% to 60%. To test this efficacy hypothesis, a sample size of 74 assessable patients provided a power of 83% with a one-sided alpha of 5%. RESULTS: A total of 79 patients were enrolled with a median follow-up of 21.0 months (interquartile range: 15.8-25.8 mo) for the primary PFS analysis. A total of 35.4% of the patients had stage IIIA, and 63.3% had stage IIIB disease. The 1-year PFS was 53.7% (95% confidence interval [CI]: 42.0%-64.0%) and the median PFS was 12.7 months (95% CI: 10.1-22.8 mo). Because 37 PFS events occurred in the first year posttreatment among the first 74 assessable patients, a 1-year PFS rate of at least 45% could not be rejected (p = 0.23). At an extended follow-up (median 32.6 mo), 37 deaths have been recorded, with a median overall survival (OS) of 38.8 months (95% CI: 26.8 mo-not estimable) and a 2-year OS rate of 63.7% (95% CI: 51.9%-73.4%). The OS of patients with stage IIIA disease was found to be significantly higher than patients with stage IIIB disease, with a 2-year OS of 81% and 56%, respectively (p = 0.037). CONCLUSIONS: PFS and OS are arithmetically higher in studies involving the same population. However, on the basis of the formal hierarchical efficacy analysis, we could not reject that the 1-year PFS rate is at least 45%.

Evolution and Clinical Impact of EGFR Mutations in Circulating Free DNA in the BELIEF Trial.

INTRODUCTION: Longitudinal evaluation of mutations in blood samples was a prespecified secondary objective in the BELIEF trial of erlotinib and bevacizumab in advanced EGFR-positive NSCLC. Here, we report the testing results and explore the correlation of EGFR status in blood with clinical outcomes. METHODS: Blood samples were prospectively collected from patients at baseline, at response evaluation, and at progression and sent to a central laboratory. Circulating free DNA was purified and EGFR mutations were analyzed with a validated real-time quantitative polymerase chain reaction assay. RESULTS: EGFR exon 19/21 mutations were detected in 55 of 91 baseline blood samples (60.4%) and correlated with a significantly worse progression-free survival: 11.4 months (95% confidence interval [CI]: 9.0-14.8 mo) for the patients who were positive versus 22.9 months (95% CI: 9.5-33.9 mo) for those who were negative (log-rank p = 0.0020). Among the 74 samples at response, exon 19/21 mutations were detected only in three samples (4.1%). In contrast, 29 of 58 patients (50.0%) were exon 19/21 positive at progression and showed a significantly worse median overall survival of 21.7 months (95% CI: 17.0-30.9 mo) compared with 37.4 months (95% CI: 22.6-53.1 mo) for those who were negative (log-rank p = 0.011). Blood samples at the three time points were available for 48 patients. Of those, among 14 exon 19/21 EGFR-negative at presentation, 13 (93%) were persistently negative for the sensitizing mutations after progression and the p.T790M could only be detected in the blood of two patients. CONCLUSIONS: Longitudinal testing of EGFR mutations in blood can offer valuable clinical information. In patients of the BELIEF study, detection of EGFR mutations in circulating free DNA at presentation was associated with shorter progression-free survival, whereas positivity at progression correlated with shorter overall survival. Finally, patients negative in blood at presentation were almost invariably negative at relapse.

A Randomized Open-Label Phase III Trial Evaluating the Addition of Denosumab to Standard First-Line Treatment in Advanced NSCLC: The European Thoracic Oncology Platform (ETOP) and European Organisation for Research and Treatment of Cancer (EORTC) SPLENDOUR Trial.

INTRODUCTION: Receptor activator of NF-kB ligand stimulates NF-kB-dependent cell signaling and acts as the primary signal for bone resorption. Retrospective analysis of a large trial comparing denosumab versus zoledronic acid in bone metastatic solid tumors suggested significant overall survival (OS) advantage for patients with lung cancer with denosumab (p = 0.01). The randomized open-label phase III SPLENDOUR trial was designed to evaluate whether the addition of denosumab to standard first-line platinum-based doublet chemotherapy improved OS in advanced NSCLC. METHODS: Patients with stage IV NSCLC were randomized in a 1:1 ratio to either chemotherapy with or without denosumab (120 mg every 3-4 wks), stratified by the presence of bone metastases (at diagnosis), Eastern Cooperative Oncology Group performance status, histology, and region. To detect an OS increase from 9 to 11.25 months (hazard ratio [HR] = 0.80), 847 OS events were required. The trial closed prematurely owing to decreasing accrual rate. RESULTS: A total of 514 patients were randomized, with 509 receiving one or more doses of the assigned treatment (chemotherapy: 252, chemotherapy-denosumab: 257). The median age was 66.1 years, 71% were men, and 59% were former smokers. Bone metastases were identified in 275 patients (53%). Median OS (95% confidence interval [CI]) was 8.7 (7.6-11.0) months in the control arm versus 8.2 (7.5-10.4) months in the chemotherapy-denosumab arm (HR = 0.96; 95% CI: 0.78-1.19; one-sided p = 0.36). For patients with bone metastasis, HR was 1.02 (95% CI: 0.77-1.35), whereas for those without, HR was 0.90 (95% CI: 0.66-1.23). Adverse events grade 3 or greater were observed in 40.9%, 5.2%, 8.7% versus 45.5%, 10.9%, 10.5% of patients. Conditional power for OS benefit was less than or equal to 10%. CONCLUSIONS: Denosumab was well-tolerated without unexpected safety concerns. There was no OS improvement for denosumab when added to chemotherapy in the intention-to-treat population and the subgroups with and without bone metastases. Our data do not provide evidence of a clinical benefit for denosumab in patients with NSCLC without bone metastases.

Inhibition of caspase-2 by a designed ankyrin repeat protein: specificity, structure, and inhibition mechanism.

Specific and potent caspase inhibitors are indispensable for the dissection of the intricate pathways leading to apoptosis. We selected a designed ankyrin repeat protein (DARPin) from a combinatorial library that inhibits caspase-2 in vitro with a subnanomolar inhibition constant and, in contrast to the peptidic caspase inhibitors, with very high specificity for this particular caspase. The crystal structure of this inhibitor (AR_F8) in complex with caspase-2 reveals the molecular basis for the specificity and, together with kinetic analyses, the allosteric mechanism of inhibition. The structure also shows a conformation of the active site that can be exploited for the design of inhibitory compounds. AR_F8 is a specific inhibitor of an initiator caspase and has the potential to help identify the function of caspase-2 in the complex biological apoptotic signaling network.

Afatinib in NSCLC With HER2 Mutations: Results of the Prospective, Open-Label Phase II NICHE Trial of European Thoracic Oncology Platform (ETOP).

INTRODUCTION: Mutations in erb-b2 receptor tyrosine kinase 2 (HER2) oncogene are observed in approximately 3% of lung adenocarcinomas or mixed tumors with adenocarcinoma component. Activity of various biologically distinct HER2 inhibitors, including the pan-HER inhibitor afatinib, has been reported in several retrospective trials or small series in advanced pretreated NSCLC with HER2 mutations. We report the first prospective evaluation of afatinib for the treatment of this molecularly defined entity. METHODS: NICHE, a single-arm phase II trial using a two-stage Simon's design, explored the potential of afatinib to control disease in pretreated patients with advanced NSCLC harboring HER2 exon 20 mutations. A total of 13 patients entered the trial and were treated with afatinib 40 mg/day until tumor progression or lack of tolerability. RESULTS: The first-stage stopping boundary was crossed when five of nine patients did not achieve disease control at 12 weeks. The accrual into the trial was stopped with total 13 patients enrolled, with 7 (53.8%) achieving disease control at 12 weeks. Except for 1 patient with early death, progression was documented for all patients, with median progression-free survival of 15.9 weeks (95% confidence interval: 6.0-35.4), and median overall survival of 56.0 weeks (95% confidence interval: 16.3- upper limit not estimable). The toxicity profile was in the expected range. CONCLUSIONS: Afatinib did not show the expected potential for disease control in NSCLC. However, more than half of the patients in the full cohort achieved disease control at 12 weeks.

Design, construction, and characterization of a second-generation DARP in library with reduced hydrophobicity.

Designed ankyrin repeat proteins (DARPins) are well-established binding molecules based on a highly stable nonantibody scaffold. Building on 13 crystal structures of DARPin-target complexes and stability measurements of DARPin mutants, we have generated a new DARPin library containing an extended randomized surface. To counteract the enrichment of unspecific hydrophobic binders during selections against difficult targets containing hydrophobic surfaces such as membrane proteins, the frequency of apolar residues at diversified positions was drastically reduced and substituted by an increased number of tyrosines. Ribosome display selections against two human caspases and membrane transporter AcrB yielded highly enriched pools of unique and strong DARPin binders which were mainly monomeric. We noted a prominent enrichment of tryptophan residues during binder selections. A crystal structure of a representative of this library in complex with caspase-7 visualizes the key roles of both tryptophans and tyrosines in providing target contacts. These aromatic and polar side chains thus substitute the apolar residues valine, leucine, isoleucine, methionine, and phenylalanine of the original DARPins. Our work describes biophysical and structural analyses required to extend existing binder scaffolds and simplifies an existing protocol for the assembly of highly diverse synthetic binder libraries.

Selective and sensitive monitoring of caspase-1 activity by a novel bioluminescent activity-based probe.

The role of caspase-1 in inflammation has been studied intensely over recent years. However, the research of caspase-1 has remained difficult mainly due to the lack of sensitive and selective tools to monitor not only its abundance but also its activity. Here we present a bioluminescent activity-based probe (ABP) for caspase-1, developed by the Reverse Design concept, where chemically optimized protease inhibitors are turned into selective substrate ABPs. The probe exhibits excellent selectivity for caspase-1 and ∼1000-fold increase in sensitivity compared to available fluorogenic peptidic caspase-1 substrates. Moreover, we have been able to monitor and quantify specific caspase-1 activity directly in cell lysates. The activity correlated well with processing of prointerleukin-1ß and prointerleukin-18 in phorbol 12-myristate 13-acetate (PMA)-stimulated cells. A detectable caspase-1 activity was present also in nonstimulated cells, consistent with processing of constitutively expressed prointerleukin-18.

The crystal structure of human pyrin b30.2 domain: implications for mutations associated with familial Mediterranean fever.

The inherited autoinflammatory syndrome familial Mediterranean fever (FMF) is characterized by recurrent episodes of fever, which are independent of any bacterial or viral infections. This disease is associated with point mutations in the mefv gene product pyrin. Although the precise molecular functions of pyrin are unknown, it seems to be involved in the maturation and secretion of interleukin-1beta. Approximately two thirds of all FMF-associated mutations cluster in the C-terminal B30.2 domain of pyrin. To investigate the molecular consequences of FMF-associated mutations, we determined the crystal structure of the pyrin B30.2 domain at 1.35-A resolution. The comparison with other B30.2/ligand complex structures revealed a shallow cavity, which seems to be involved in binding the pyrin ligand. The bottom of this cavity is covered mainly with hydrophobic amino acids, suggesting that pyrin recognizes its ligand by hydrophobic contacts and surface complementarities. FMF-associated mutations cluster around two sites on the B30.2 surface. Approximately two thirds, including those mutations with the most severe disease outcomes, are observed in the vicinity of the predicted peptide binding site, suggesting that they will have a direct impact on ligand binding. A second mutational hot spot was observed on the opposite side of the B30.2 domain in the neighbourhood of its artificial N-terminus. Although most FMF-associated mutations are solvent exposed, several will modify the main-chain conformation of loops. The experimental crystal structure of the pyrin B30.2 domain serves as a basis for an accurate modelling of these mutations.