Mixed responses to first-line alectinib in non-small cell lung cancer patients with rare ALK gene fusions: A case series and literature review

Anaplastic lymphoma kinase (ALK) fusion is a well-defined biomarker for ALK tyrosine kinase inhibitors (TKIs) treatment in non-small cell lung cancer (NSCLC). Alectinib, a second-generation ALK-TKI, has been shown to have significantly longer progression-free survival (PFS) than first-generation ALK inhibitors in untreated ALK-rearranged NSCLC patients. However, its clinical efficacy on rare ALK fusions remains unclear. Herein, two advanced NSCLC patients received first-line alectinib treatment, given their positive ALK fusion status as determined by immunohistochemistry (IHC) testing results. Patients showed limited clinical response (PFS: 4 months) and primary resistance to alectinib respectively. Molecular profiling using next-generation sequencing (NGS) further revealed a striatin (STRN)-ALK fusion in the first patient accompanied by MET amplification, and a LIM domain only protein 7 (LMO7)-ALK fusion in another patient without any other known oncogenic alterations. Both patients demonstrated improved survival after they switched to second-line crizotinib (PFS: 11 months) and ensartinib (PFS: 18 months), respectively, up till the last follow-up assessment. In conclusion, the clinical efficacy of ALK-TKIs including alectinib for lung cancer with uncommon ALK gene fusions is still under evaluation. This study and literature review results showed mixed responses to alectinib in NSCLC patients who harboured rare ALK fusions. Comprehensive molecular profiling of tumour is thus strongly warranted for precise treatment strategies.     

miR‐126‐5p enhances radiosensitivity of lung adenocarcinoma cells by inhibiting EZH2 via the KLF2/BIRC axis

Radiotherapy is a common method for the treatment of lung adenocarcinoma, but it often fails due to the relative non‐susceptibility of lung adenocarcinoma cells to radiation. We aimed to discuss the related mechanisms by which miR‐126‐5p might mediate radiosensitivity of lung adenocarcinoma cells. The binding affinity between miR‐126‐5p and EZH2 and between KLF2 and BIRC5 was identified using multiple assays. A549 and H1650 cells treated with X‐ray were transfected with miR‐126‐5p mimic/inhibitor, oe‐EZH2, or si‐KLF2 to detect cell biological functions and radiosensitivity. Finally, lung adenocarcinoma nude mouse models were established. miR‐126‐5p and KLF2 were poorly expressed, while EZH2 and BIRC5 were upregulated in lung adenocarcinoma tissues and cells. miR‐126‐5p targeted EZH2 to promote the KLF2 expression so as to inhibit BIRC5 activation. Both in vitro and in vivo experiments verified that elevated miR‐126‐5p inhibited cell migration and promoted apoptosis to enhance the sensitivity of lung adenocarcinoma cells to radiotherapy via the EZH2/KLF2/BIRC5 axis. Collectively, miR‐126‐5p downregulated EZH2 to facilitate the sensitivity of lung adenocarcinoma cells to radiotherapy via KLF2/BIRC5.     

ADAM10 and ADAM17 promote SARS‐CoV‐2 cell entry and spike protein‐mediated lung cell fusion

The severe‐acute‐respiratory‐syndrome‐coronavirus‐2 (SARS‐CoV‐2) is the causative agent of COVID‐19, but host cell factors contributing to COVID‐19 pathogenesis remain only partly understood. We identify the host metalloprotease ADAM17 as a facilitator of SARS‐CoV‐2 cell entry and the metalloprotease ADAM10 as a host factor required for lung cell syncytia formation, a hallmark of COVID‐19 pathology. ADAM10 and ADAM17, which are broadly expressed in the human lung, cleave the SARS‐CoV‐2 spike protein (S) in vitro, indicating that ADAM10 and ADAM17 contribute to the priming of S, an essential step for viral entry and cell fusion. ADAM protease‐targeted inhibitors severely impair lung cell infection by the SARS‐CoV‐2 variants of concern alpha, beta, delta, and omicron and also reduce SARS‐CoV‐2 infection of primary human lung cells in a TMPRSS2 protease‐independent manner. Our study establishes ADAM10 and ADAM17 as host cell factors for viral entry and syncytia formation and defines both proteases as potential targets for antiviral drug development.     

Method for quick DNA barcode reference library construction

DNA barcoding has become one of the most important techniques in plant species identification. Successful application of this technology is dependent on the availability of reference database of high species coverage. Unfortunately, there are experimental and data processing challenges to construct such a library within a short time. Here, we present our solutions to these challenges. We sequenced six conventional DNA barcode fragments (ITS1, ITS2, matK1, matK2, rbcL1, and rbcL2) of 380 flowering plants on next‐generation sequencing (NGS) platforms (Illumina Hiseq 2500 and Ion Torrent S5) and the Sanger sequencing platform. After comparing the sequencing depths, read lengths, base qualities, and base accuracies, we conclude that Illumina Hiseq2500 PE250 run is suitable for conventional DNA barcoding. We developed a new “Cotu” method to create consensus sequences from NGS reads for longer output sequences and more reliable bases than the other three methods. Step‐by‐step instructions to our method are provided. By using high‐throughput machines (PCR and NGS), labeling PCR, and the Cotu method, it is possible to significantly reduce the cost and labor investments for DNA barcoding. A regional or even global DNA barcoding reference library with high species coverage is likely to be constructed in a few years.     

Phosphodiesterase 4A confers resistance to PGE2‐mediated suppression in CD25+/CD54+ NK cells

Inadequate persistence of tumor‐infiltrating natural killer (NK) cells is associated with poor prognosis in cancer patients. The solid tumor microenvironment is characterized by the presence of immunosuppressive factors, including prostaglandin E2 (PGE2), that limit NK cell persistence. Here, we investigate if the modulation of the cytokine environment in lung cancer with IL‐2 or IL‐15 renders NK cells resistant to suppression by PGE2. Analyzing Cancer Genome Atlas (TCGA) data, we found that high NK cell gene signatures correlate with significantly improved overall survival in patients with high levels of the prostaglandin E synthase (PTGES). In vitro, IL‐15, in contrast to IL‐2, enriches for CD25⁺/CD54⁺ NK cells with superior mTOR activity and increased expression of the cAMP hydrolyzing enzyme phosphodiesterase 4A (PDE4A). Consequently, this distinct population of NK cells maintains their function in the presence of PGE2 and shows an increased ability to infiltrate lung adenocarcinoma tumors in vitro and in vivo. Thus, strategies to enrich CD25⁺/CD54⁺ NK cells for adoptive cell therapy should be considered.     

A pipeline for effectively developing highly polymorphic simple sequence repeats markers based on multi‐sample genomic data

Simple sequence repeats (SSRs) are widely used genetic markers in ecology, evolution, and conservation even in the genomics era, while a general limitation to their application is the difficulty of developing polymorphic SSR markers. Next‐generation sequencing (NGS) offers the opportunity for the rapid development of SSRs; however, previous studies developing SSRs using genomic data from only one individual need redundant experiments to test the polymorphisms of SSRs. In this study, we designed a pipeline for the rapid development of polymorphic SSR markers from multi‐sample genomic data. We used bioinformatic software to genotype multiple individuals using resequencing data, detected highly polymorphic SSRs prior to experimental validation, significantly improved the efficiency and reduced the experimental effort. The pipeline was successfully applied to a globally threatened species, the brown eared‐pheasant (Crossoptilon mantchuricum), which showed very low genomic diversity. The 20 newly developed SSR markers were highly polymorphic, the average number of alleles was much higher than the genomic average. We also evaluated the effect of the number of individuals and sequencing depth on the SSR mining results, and we found that 10 individuals and ~10X sequencing data were enough to obtain a sufficient number of polymorphic SSRs, even for species with low genetic diversity. Furthermore, the genome assembly of NGS data from the optimal number of individuals and sequencing depth can be used as an alternative reference genome if a high‐quality genome is not available. Our pipeline provided a paradigm for the application of NGS technology to mining and developing molecular markers for ecological and evolutionary studies.     

Caveolin‐1 negatively regulates inflammation and fibrosis in silicosis

Inhalation of crystalline silica causes silicosis, the most common and serious occupational disease, which is characterized by progressive lung inflammation and fibrosis. Recent studies revealed the anti‐inflammatory and anti‐fibrosis role of Caveolin‐1 (Cav‐1) in lung, but this role in silicosis has not been investigated. Thus, this study evaluated Cav‐1 regulatory effects in silicosis. It was found that Cav‐1 levels were significantly reduced in the lung from silicosis patients and silicotic mice. The silicosis models were established in C57BL/6 (wild‐type) and Cav‐1 deficiency (Cav‐1 ^−/−) mice, and Cav‐1 ^−/− mice displayed wider alveolar septa, increased collagen deposition and more silicotic nodules. The mice peritoneal‐derived macrophages were used to explore the role of Cav‐1 in silica‐induced inflammation, which plays a central role in mechanism of silicosis. Cav‐1 inhibited silica‐induced infiltration of inflammatory cells and secretion of inflammatory factors in vitro and in vivo, partly by downregulating NF‐κB pathway. Additionally, silica uptake and expression of 4‐hydroxynonenal in silicotic mice were observed, and it was found that Cav‐1 absence triggered excessive silica deposition, causing a stronger oxidative stress response. These findings demonstrate the protective effects of Cav‐1 in silica‐induced lung injury, suggesting its potential therapeutic value in silicosis.     

Genome‐wide selective detection of Mile red‐bone goat using next‐generation sequencing technology

The ecotype population of goats (Capra hircus) was created by long‐term artificial selection and natural adaptation. Mile red‐bone goat is an indigenous breed with visible red bones, and its special bone structure has received extensive attention. This study aimed to identify genetic variants and candidate genes associated with specific bone phenotypes using next‐generation sequencing technology (NGS). The results revealed that 31,828,206 single nucleotide polymorphisms (SNPs) were obtained from 72 goats (20 Mile red‐bone goats and 52 common goats) by NGS. A total of 100 candidate genes were identified on the basis top 1% window interaction from nucleotide diversity (π), π ratio (π _A/π _B), and pairwise fixation index (F _ST). Exactly 77 known signaling pathways were enriched. Specifically, three coding genes (NMNAT2, LOC102172983, and PNLIP) were annotated in the vitamin metabolism signaling pathways, and NCF2 was annotated to the osteoclast (OC) differentiation pathway. Furthermore, 5862 reliable copy number variations (CNVs) were obtained, and 14 and 24 genes were annotated with the top 1‰ CNV based on F _ST (>0.490) and V _ST (>0.527), respectively. Several pathways related to bone development and metabolism of exogenous substances in vivo, including calcium signaling pathway, OC differentiation, and glycerophospholipid metabolism, were annotated. Specifically, six genes from 19 candidate CNVs, which were obtained by interaction of the top 1‰ CNVs with F _ST and V _ST, were annotated to mucin‐type O‐glycan biosynthesis and metabolic pathways. Briefly, the results implied that pseudopurpurin and specific genetic variants work together to contribute to the red‐bone color and specific bone structure of Mile red‐bone goat. This study is helpful to understanding the genetic basis of the unique bone phenotype of Mile red‐bone goats.     

Novel long non‐coding RNA CYB561‐5 promotes aerobic glycolysis and tumorigenesis by interacting with basigin in non‐small cell lung cancer

Abnormally expressed long non‐coding RNAs (lncRNAs) have been recognized as potential diagnostic biomarkers or therapeutic targets in non‐small cell lung cancer (NSCLC). The role of the novel lnc‐CYB561‐5 in NSCLC and its specific biological activity remain unknown. In this study, lncRNAs highly expressed in NSCLC tissue samples compared with paired adjacent normal tissue samples and atypical adenomatous hyperplasia were identified by RNA‐seq analysis. Lnc‐CYB561‐5 is highly expressed in human NSCLC and is associated with a poor prognosis in lung adenocarcinoma. In vivo, downregulation of lnc‐CYB561‐5 significantly decreases tumour growth and metastasis. In vitro, lnc‐CYB561‐5 knockdown treatment inhibits cell migration, invasion and proliferation ability, as well as glycolysis rates. In addition, RNA pulldown and RNA immunoprecipitation (RIP) assays show that basigin (Bsg) protein interacts with lnc‐CYB561‐5. Overall, this study demonstrates that lnc‐CYB561‐5 is an oncogene in NSCLC, which is involved in the regulation of cell proliferation and metastasis. Lnc‐CYB561‐5 interacts with Bsg to promote the expression of Hk2 and Pfk1 and further lead to metabolic reprogramming of NSCLC cells.     

Opposing activities of IFITM proteins in SARS‐CoV‐2 infection

Interferon‐induced transmembrane proteins (IFITMs) restrict infections by many viruses, but a subset of IFITMs enhance infections by specific coronaviruses through currently unknown mechanisms. We show that SARS‐CoV‐2 Spike‐pseudotyped virus and genuine SARS‐CoV‐2 infections are generally restricted by human and mouse IFITM1, IFITM2, and IFITM3, using gain‐ and loss‐of‐function approaches. Mechanistically, SARS‐CoV‐2 restriction occurred independently of IFITM3 S‐palmitoylation, indicating a restrictive capacity distinct from reported inhibition of other viruses. In contrast, the IFITM3 amphipathic helix and its amphipathic properties were required for virus restriction. Mutation of residues within the IFITM3 endocytosis‐promoting YxxФ motif converted human IFITM3 into an enhancer of SARS‐CoV‐2 infection, and cell‐to‐cell fusion assays confirmed the ability of endocytic mutants to enhance Spike‐mediated fusion with the plasma membrane. Overexpression of TMPRSS2, which increases plasma membrane fusion versus endosome fusion of SARS‐CoV‐2, attenuated IFITM3 restriction and converted amphipathic helix mutants into infection enhancers. In sum, we uncover new pro‐ and anti‐viral mechanisms of IFITM3, with clear distinctions drawn between enhancement of viral infection at the plasma membrane and amphipathicity‐based mechanisms used for endosomal SARS‐CoV‐2 restriction.     

SARS‐CoV‐2 Alpha,Beta, and Delta variants display enhanced Spike‐mediated syncytia formation

Severe COVID‐19 is characterized by lung abnormalities, including the presence of syncytial pneumocytes. Syncytia form when SARS‐CoV‐2 spike protein expressed on the surface of infected cells interacts with the ACE2 receptor on neighboring cells. The syncytia forming potential of spike variant proteins remain poorly characterized. Here, we first assessed Alpha (B.1.1.7) and Beta (B.1.351) spread and fusion in cell cultures, compared with the ancestral D614G strain. Alpha and Beta replicated similarly to D614G strain in Vero, Caco‐2, Calu‐3, and primary airway cells. However, Alpha and Beta formed larger and more numerous syncytia. Variant spike proteins displayed higher ACE2 affinity compared with D614G. Alpha, Beta, and D614G fusion was similarly inhibited by interferon‐induced transmembrane proteins (IFITMs). Individual mutations present in Alpha and Beta spikes modified fusogenicity, binding to ACE2 or recognition by monoclonal antibodies. We further show that Delta spike also triggers faster fusion relative to D614G. Thus, SARS‐CoV‐2 emerging variants display enhanced syncytia formation.     

Clinical and prognostic implications of an immune‐related risk model based on TP53 status in lung adenocarcinoma

TP53 mutation is the most widespread mutation in lung adenocarcinoma (LUAD). Meanwhile, p53 (encoded by TP53) has recently been implicated in immune responses. However, it is still unknown whether TP53 mutation remodels the tumour microenvironment to influence tumour progression and prognosis in LUAD. In this study, we developed a 6‐gene immune‐related risk model (IRM) to predict the survival of patients with LUAD in The Cancer Genome Atlas (TCGA) cohort based on TP53 status, and the predictive ability was confirmed in 2 independent cohorts. TP53 mutation led to a decreased immune response in LUAD. Further analysis revealed that patients in the high‐index group had observably lower relative infiltration of memory B cells and regulatory T cells and significantly higher relative infiltration of neutrophils and resting memory CD4⁺ T cells. Additionally, the IRM index positively correlated with the expression of critical immune checkpoint genes, including PDCD1 (encoding PD‐1) and CD274 (encoding PD‐L1), which was validated in the Nanjing cohort. Furthermore, as an independent prognostic factor, the IRM index was used to establish a nomogram for clinical application. In conclusion, this IRM may serve as a powerful prognostic tool to further optimize LUAD immunotherapy.     

Non‐canonical autophagy functions of ATG16L1 in epithelial cells limit lethal infection by influenza A virus

Influenza A virus (IAV) and SARS‐CoV‐2 (COVID‐19) cause pandemic infections where cytokine storm syndrome and lung inflammation lead to high mortality. Given the high social and economic cost of respiratory viruses, there is an urgent need to understand how the airways defend against virus infection. Here we use mice lacking the WD and linker domains of ATG16L1 to demonstrate that ATG16L1‐dependent targeting of LC3 to single‐membrane, non‐autophagosome compartments – referred to as non‐canonical autophagy – protects mice from lethal IAV infection. Mice with systemic loss of non‐canonical autophagy are exquisitely sensitive to low‐pathogenicity IAV where extensive viral replication throughout the lungs, coupled with cytokine amplification mediated by plasmacytoid dendritic cells, leads to fulminant pneumonia, lung inflammation and high mortality. IAV was controlled within epithelial barriers where non‐canonical autophagy reduced IAV fusion with endosomes and activation of interferon signalling. Conditional mouse models and ex vivo analysis showed that protection against IAV infection of lung was independent of phagocytes and other leucocytes. This establishes non‐canonical autophagy in airway epithelial cells as a novel innate defence that restricts IAV infection and lethal inflammation at respiratory surfaces.     

Sensing of mycobacterial arabinogalactan by galectin‐9 exacerbates mycobacterial infection

Mycobacterial arabinogalactan (AG) is an essential cell wall component of mycobacteria and a frequent structural and bio‐synthetical target for anti‐tuberculosis (TB) drug development. Here, we report that mycobacterial AG is recognized by galectin‐9 and exacerbates mycobacterial infection. Administration of AG‐specific aptamers inhibits cellular infiltration caused by Mycobacterium tuberculosis (Mtb) or Mycobacterium bovis BCG, and moderately increases survival of Mtb‐infected mice or Mycobacterium marinum‐infected zebrafish. AG interacts with carbohydrate recognition domain (CRD) 2 of galectin‐9 with high affinity, and galectin‐9 associates with transforming growth factor β‐activated kinase 1 (TAK1) via CRD2 to trigger subsequent activation of extracellular signal‐regulated kinase (ERK) as well as induction of the expression of matrix metalloproteinases (MMPs). Moreover, deletion of galectin‐9 or inhibition of MMPs blocks AG‐induced pathological impairments in the lung, and the AG‐galectin‐9 axis aggravates the process of Mtb infection in mice. These results demonstrate that AG is an important virulence factor of mycobacteria and galectin‐9 is a novel receptor for Mtb and other mycobacteria, paving the way for the development of novel effective TB immune modulators.     

Insect cell expression and purification of recombinant SARS‐COV‐2 spike proteins that demonstrate ACE2 binding

The COVID‐19 pandemic caused by SARS‐CoV‐2 infection has led to socio‐economic shutdowns and the loss of over 5 million lives worldwide. There is a need for the identification of therapeutic targets to treat COVID‐19. SARS‐CoV‐2 spike is a target of interest for the development of therapeutic targets. We developed a robust SARS‐CoV‐2 S spike expression and purification protocol from insect cells and studied four recombinant SARS‐CoV‐2 spike protein constructs based on the original SARS‐CoV‐2 sequence using a baculovirus expression system: a spike protein receptor‐binding domain that includes the SD1 domain (RBD) coupled to a fluorescent tag (S‐RBD‐eGFP), spike ectodomain coupled to a fluorescent tag (S‐Ecto‐eGFP), spike ectodomain with six proline mutations and a foldon domain (S‐Ecto‐HexaPro(+F)), and spike ectodomain with six proline mutations without the foldon domain (S‐Ecto‐HexaPro(‐F)). We tested the yield of purified protein expressed from the insect cell lines Spodoptera frugiperda (Sf9) and Trichoplusia ni (Tni) and compared it to previous research using mammalian cell lines to determine changes in protein yield. We demonstrated quick and inexpensive production of functional glycosylated spike protein of high purity capable of recognizing and binding to the angiotensin converting enzyme 2 (ACE2) receptor. To further confirm functionality, we demonstrate binding of eGFP fused construct of the spike ectodomain (S‐Ecto‐eGFP) to surface ACE2 receptors on lung epithelial cells by flow cytometry analysis and show that it can be decreased by means of receptor manipulation (blockade or downregulation).     

Attenuation of SARS‐CoV‐2 replication and associated inflammation by concomitant targeting of viral and host cap 2'‐O‐ribose methyltransferases

The SARS‐CoV‐2 infection cycle is a multistage process that relies on functional interactions between the host and the pathogen. Here, we repurposed antiviral drugs against both viral and host enzymes to pharmaceutically block methylation of the viral RNA 2''‐O‐ribose cap needed for viral immune escape. We find that the host cap 2''‐O‐ribose methyltransferase MTr1 can compensate for loss of viral NSP16 methyltransferase in facilitating virus replication. Concomitant inhibition of MTr1 and NSP16 efficiently suppresses SARS‐CoV‐2 replication. Using in silico target‐based drug screening, we identify a bispecific MTr1/NSP16 inhibitor with anti‐SARS‐CoV‐2 activity in vitro and in vivo but with unfavorable side effects. We further show antiviral activity of inhibitors that target independent stages of the host SAM cycle providing the methyltransferase co‐substrate. In particular, the adenosylhomocysteinase (AHCY) inhibitor DZNep is antiviral in in vitro, in ex vivo, and in a mouse infection model and synergizes with existing COVID‐19 treatments. Moreover, DZNep exhibits a strong immunomodulatory effect curbing infection‐induced hyperinflammation and reduces lung fibrosis markers ex vivo. Thus, multispecific and metabolic MTase inhibitors constitute yet unexplored treatment options against COVID‐19.     

ANKRD49 promotes the invasion and metastasis of lung adenocarcinoma via a P38/ATF‐2 signalling pathway

Lung adenocarcinoma (LUAD) is the most challenging neoplasm to treat in clinical practice. Ankyrin repeat domain 49 protein (ANKRD49) is highly expressed in several carcinomas; however, its pattern of expression and role in LUAD are not known. Tissue microarrays, immunohistochemistry, χ ² test, Spearman correlation analysis, Kaplan–Meier, log‐rank test, and Cox''s proportional hazard model were used to analyse the clinical cases. The effect of ANKRD49 on the LUAD was investigated using CCK‐8, clonal formation, would healing, transwell assays, and nude mice experiment. Expressions of ANKRD49 and its associated downstream protein molecules were verified by real‐time PCR, Western blot, immunohistochemistry, and/or immunofluorescence analyses. ANKRD49 expression was highly elevated in LUAD. The survival rate and Cox''s modelling analysis indicated that there may be an independent prognostic indicator for LUAD patients. We also found that ANKRD49 promoted the invasion and migration in both in in vitro and in vivo assays, through upregulating matrix metalloproteinase (MMP)‐2 and MMP‐9 activities via the P38/ATF‐2 signalling pathway Our findings suggest that ANKRD49 is a latent biomarker for evaluating LUAD prognosis and promotes the metastasis of A549 cells via upregulation of MMP‐2 and MMP‐9 in a P38/ATF‐2 pathway‐dependent manner.