Healthy aging depends on removal of damaged cellular material that is in part mediated by autophagy. The nutritional status of cells affects both aging and autophagy through as-yet-elusive metabolic ...circuitries. Here, we show that nucleocytosolic acetyl-coenzyme A (AcCoA) production is a metabolic repressor of autophagy during aging in yeast. Blocking the mitochondrial route to AcCoA by deletion of the CoA-transferase ACH1 caused cytosolic accumulation of the AcCoA precursor acetate. This led to hyperactivation of nucleocytosolic AcCoA-synthetase Acs2p, triggering histone acetylation, repression of autophagy genes, and an age-dependent defect in autophagic flux, culminating in a reduced lifespan. Inhibition of nutrient signaling failed to restore, while simultaneous knockdown of ACS2 reinstated, autophagy and survival of ach1 mutant. Brain-specific knockdown of Drosophila AcCoA synthetase was sufficient to enhance autophagic protein clearance and prolong lifespan. Since AcCoA integrates various nutrition pathways, our findings may explain diet-dependent lifespan and autophagy regulation.
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•Acetyl-CoA (AcCoA) metabolism regulates autophagy during aging•Autophagy regulation by AcCoA metabolism acts downstream of nutrient signaling•Brain-specific knockdown of Drosophila AcCoA synthetase prolongs lifespan•Histone point mutations permanently activate autophagy during aging
Autophagy plays a crucial role in healthy aging. By blocking mitochondrial AcCoA production, Eisenberg et al. show that accumulation of nucleocytosolic AcCoA inhibits autophagy and reduces lifespan through a conserved epigenetic mechanism involving histone acetylation of specific autophagy genes in yeast and flies.
Different mechanisms mediate the toxicity of RNA. Genomic retroviral mRNA hijacks infected host cell factors to enable virus replication. The viral genomic RNA of the human immunodeficiency virus ...(HIV) encompasses nine genes encoding in less than 10 kb all proteins needed for replication in susceptible host cells. To do so, the genomic RNA undergoes complex alternative splicing to facilitate the synthesis of the structural, accessory, and regulatory proteins. However, HIV strongly relies on the host cell machinery recruiting cellular factors to complete its replication cycle. Antiretroviral therapy (ART) targets different steps in the cycle, preventing disease progression to the acquired immunodeficiency syndrome (AIDS). The comprehension of the host immune system interaction with the virus has fostered the development of a variety of vaccine platforms. Despite encouraging provisional results in vaccine trials, no effective vaccine has been developed, yet. However, novel promising vaccine platforms are currently under investigation.
Circulating tumor cells (CTCs) hold great potential to answer key questions of how non-small cell lung cancer (NSCLC) evolves and develops resistance upon anti-PD-1/PD-L1 treatment. Currently, their ...clinical utility in NSCLC is compromised by a low detection rate with the established, Food and Drug Administration (FDA)-approved, EpCAM-based CellSearch
System. We tested an epitope-independent method (Parsortix
system) and utilized it to assess PD-L1 expression of CTCs from NSCLC patients. We prospectively collected 127 samples, 97 of which were analyzed with the epitope-independent system in comparison to the CellSearch system. CTCs were determined by immunocytochemistry as intact, nucleated, CD45
, pankeratins (K)
cells. PD-L1 status of CTCs was evaluated from 89 samples. With the epitope-independent system, ≥1 CTC per blood sample was detected in 59 samples (61%) compared to 31 samples (32%) with the EpCAM-based system. Upon PD-L1 staining, 47% of patients harbored only PD-L1
CTCs, 47% had PD-L1
and PD-L1
CTCs, and only 7% displayed exclusively PD-L1
CTCs. The percentage of PD-L1
CTCs did not correlate with the percentage of PD-L1
in biopsies determined by immunohistochemistry (
= 0.179). Upon disease progression, all patients showed an increase in PD-L1
CTCs, while no change or a decrease in PD-L1
CTCs was observed in responding patients (
= 11;
= 0.001). Our data show a considerable heterogeneity in the PD-L1 status of CTCs from NSCLC patients. An increase of PD-L1
CTCs holds potential to predict resistance to PD-1/PD-L1 inhibitors.
Osimertinib has become the preferred first-line therapy for epidermal growth factor receptor (EGFR) mutation-positive metastatic non-small cell lung cancer (NSCLC) in recent years. Originally, it was ...approved for second-line treatment after epidermal growth factor receptor EGFR tyrosine kinase inhibitors (TKIs) of the first and second generations had failed and EGFR T790M had emerged as a mode of resistance. Osimertinib itself provokes a wide array of on- and off-target molecular alterations that can limit therapeutic success. Liquid biopsy ctDNA (circulating tumor DNA) analysis by hybrid capture (HC) next-generation sequencing (NGS) can help to identify alterations in a minimally invasive way and allows for the detection of common as well as rare resistance alterations. We describe a young female patient who was initially diagnosed with metastatic EGFR L858R-positive NSCLC. She received EGFR TKI therapy at different timepoints during the course of the disease and developed sequential EGFR resistance alterations (EGFR T790M and C797S). In the course of her disease, resistance alteration became undetectable, and the tumor was successfully rechallenged with the original first-generation EGFR TKI as well as osimertinib and altogether showed prolonged response despite a prognostically negative TP53 alteration. To date, the patient has been alive for more than seven years, though initially diagnosed with a heavy metastatic burden.
HER2-targeted therapy is currently the subject of several studies in lung cancer and other solid tumors using either tyrosine kinase inhibitors (TKI) or targeted-antibody-drug conjugates. We describe ...a 61-year-old female patient with HER2 mutated adenocarcinoma of the lungs who received chemo-immunotherapy, followed by trastuzumab deruxtecan (T-DXd) and third-line Ramucirumab/Docetaxel at disease progression. Plasma ctDNA monitoring was obtained at 12 timepoints during therapy and revealed HER2 mutation allele frequencies that corresponded to the clinical course of disease. HER2-targeted T-DXd therapy resulted in a profound clinical response and may be an option for NSCLC patients carrying an activated HER2 mutation. Longitudinal liquid biopsy quantification of the underlying driver alteration can serve as a powerful diagnostic tool to monitor course of therapy.
The worldwide approval of the combination maintenance therapy of olaparib and bevacizumab in advanced high-grade serous ovarian cancer requires complex molecular diagnostic assays that are ...sufficiently robust for the routine detection of driver mutations in homologous recombination repair (HRR) genes and genomic instability (GI), employing formalin-fixed (FFPE) paraffin-embedded tumor samples without matched normal tissue. We therefore established a DNA-based hybrid capture NGS assay and an associated bioinformatic pipeline that fulfils our institution's specific needs. The assay´s target regions cover the full exonic territory of relevant cancer-related genes and HRR genes and more than 20,000 evenly distributed single nucleotide polymorphism (SNP) loci to allow for the detection of genome-wide allele specific copy number alterations (CNA). To determine GI status, we implemented an %CNA score that is robust across a broad range of tumor cell content (25-85%) often found in routine FFPE samples. The assay was established using high-grade serous ovarian cancer samples for which
and
mutation status as well as Myriad MyChoice homologous repair deficiency (HRD) status was known. The NOGGO (Northeastern German Society for Gynecologic Oncology) GIS (GI-Score) v1 assay was clinically validated on more than 400 samples of the ENGOT PAOLA-1 clinical trial as part of the European Network for Gynaecological Oncological Trial groups (ENGOT) HRD European Initiative. The "NOGGO GIS v1 assay" performed using highly robust hazard ratios for progression-free survival (PFS) and overall survival (OS), as well a significantly lower dropout rate than the Myriad MyChoice clinical trial assay supporting the clinical utility of the assay. We also provide proof of a modular and scalable routine diagnostic method, that can be flexibly adapted and adjusted to meet future clinical needs, emerging biomarkers, and further tumor entities.
Since 2009, several first, second, and third generation EGFR tyrosine kinase inhibitors (TKI) have been approved for targeted treatment of EGFR mutated metastatic non-small lung cancer (NSCLC). A ...vast majority of patients is improving quickly on treatment; however, resistance is inevitable and typically occurs after one year for TKI of the first and second generation. Osimertinib, a third generation TKI, has recently been approved for first line treatment in the palliative setting and is expected to become approved for the adjuvant setting as well. Progression-free survival (PFS) under osimertinib is superior to its predecessors but its spectrum of resistance alterations appears significantly more diverse compared to first and second generation EGFR TKI. As resistance mechanisms to osimertinib are therapeutically targetable in some cases, it is important to comprehensively test for molecular alterations in the relapse scenario. Liquid biopsy may be advantageous over tissue analysis as it has the potential to represent tumor heterogeneity and clonal diversification. We have previously shown high concordance of hybrid capture (HC) based next generation sequencing (NGS) in liquid biopsy versus solid tumor biopsies. In this study, we now present real-word data from 56 patients with metastatic NSCLC that were tested by liquid biopsy at the time of disease progression on mostly second line treated osimertinib treatment. We present examples of single and multiple TKI resistance mechanisms, including mutations in multiple pathways, copy number changes and rare fusions of RET, ALK, FGFR3 and BRAF. In addition, we present the added value of HC based NGS to reveal polyclonal resistance development at the DNA level encoding multiple EGFR C797S and PIK3CA mutations.
To date, the establishment of high-titer stable viral packaging cells (VPCs) at large scale for gene therapeutic applications is very time- and cost-intensive. Here we report the establishment of ...three human suspension 293-F-derived ecotropic MLV-based VPCs. The classic stable transfection of an EGFP-expressing transfer vector resulted in a polyclonal VPC pool that facilitated cultivation in shake flasks of 100 mL volumes and yielded high functional titers of more than 1 × 10
transducing units/mL (TU/mL). When the transfer vector was flanked by transposon terminal inverted repeats (TIRs) and upon co-transfection of a plasmid encoding for the transposase, productivities could be slightly elevated to more than 3 × 10
TU/mL. In contrast and using mRNA encoding for the transposase, as a proof of concept, productivities were drastically improved by more than ten-fold exceeding 5 × 10
TU/mL. In addition, these VPC pools were generated within only 3 weeks. The production volume was successfully scaled up to 500 mL employing a stirred-tank bioreactor (STR). We anticipate that the stable transposition of transfer vectors employing transposase transcripts will be of utility for the future establishment of high-yield VPCs producing pseudotype vector particles with a broader host tropism on a large scale.
In recent years, Non-small cell lung cancer (NSCLC) has evolved into a prime example for precision oncology with multiple FDA-approved “precision” drugs. For the majority of NSCLC lacking targetable ...genetic alterations, immune checkpoint inhibition (ICI) has become standard of care in first-line treatment or beyond. PD-L1 tumor expression represents the only approved predictive biomarker for PD-L1/PD-1 checkpoint inhibition by therapeutic antibodies. Since PD-L1-negative or low-expressing tumors may also respond to ICI, additional factors are likely to contribute in addition to PD-L1 expression. Tumor mutation burden (TMB) has emerged as a potential candidate; however, it is the most complex biomarker so far and might represent a challenge for routine diagnostics. We therefore established a hybrid capture (HC) next-generation sequencing (NGS) assay that covers all oncogenic driver alterations as well as TMB and validated TMB values by correlation with the assay (F1CDx) used for the CheckMate 227 study. Results of the first consecutive 417 patients analyzed in a routine clinical setting are presented. Data show that fast reliable comprehensive diagnostics including TMB and targetable alterations are obtained with a short turn-around time. Thus, even complex biomarkers can easily be implemented in routine practice to optimize treatment decisions for advanced NSCLC.
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