Structures of ISCth4 transpososomes reveal the role of asymmetry in copy-out/paste-in DNA transposition.

Copy-out/paste-in transposition is a major bacterial DNA mobility pathway. It contributes significantly to the emergence of antibiotic resistance, often by upregulating expression of downstream genes upon integration. Unlike other transposition pathways, it requires both asymmetric and symmetric strand transfer steps. Here, we report the first structural study of a copy-out/paste-in transposase and demonstrate its ability to catalyze all pathway steps in vitro. X-ray structures of ISCth4 transposase, a member of the IS256 family of insertion sequences, bound to DNA substrates corresponding to three sequential steps in the reaction reveal an unusual asymmetric dimeric transpososome. During transposition, an array of N-terminal domains binds a single transposon end while the catalytic domain moves to accommodate the varying substrates. These conformational changes control the path of DNA flanking the transposon end and the generation of DNA-binding sites. Our results explain the asymmetric outcome of the initial strand transfer and show how DNA binding is modulated by the asymmetric transposase to allow the capture of a second transposon end and to integrate a circular intermediate.

LINE-1 repression in Epstein-Barr virus-associated gastric cancer through viral-host genome interaction.

Long INterspersed Element 1 (LINE-1 or L1) acts as a major remodeling force in genome regulation and evolution. Accumulating evidence shows that virus infection impacts L1 expression, potentially impacting host antiviral response and diseases. The underlying regulation mechanism is unclear. Epstein-Barr virus (EBV), a double-stranded DNA virus linked to B-cell and epithelial malignancies, is known to have viral-host genome interaction, resulting in transcriptional rewiring in EBV-associated gastric cancer (EBVaGC). By analyzing publicly available datasets from the Gene Expression Omnibus (GEO), we found that EBVaGC has L1 transcriptional repression compared with EBV-negative gastric cancer (EBVnGC). More specifically, retrotransposition-associated young and full-length L1s (FL-L1s) were among the most repressed L1s. Epigenetic alterations, especially increased H3K9me3, were observed on FL-L1s. H3K9me3 deposition was potentially attributed to increased TASOR expression, a key component of the human silencing hub (HUSH) complex for H3K9 trimethylation. The 4C- and HiC-seq data indicated that the viral DNA interacted in the proximity of the TASOR enhancer, strengthening the loop formation between the TASOR enhancer and its promoter. These results indicated that EBV infection is associated with increased H3K9me3 deposition, leading to L1 repression. This study uncovers a regulation mechanism of L1 expression by chromatin topology remodeling associated with viral-host genome interaction in EBVaGC.

Repurposing Axl Kinase Inhibitors for the Treatment of Respiratory Syncytial Virus Infection.

Respiratory syncytial virus (RSV) infection persists as a common pathogen of pulmonary infection in infants and in the elderly with high morbidity and mortality. However, no specific therapeutics are available. Axl, a member of the TAM (Tyro3, Axl, and Mertk) family receptor kinases, is a pleiotropic inhibitor of the innate immune response and functions as a negative regulator of interferon pathway activation. In this report, we investigated Axl inhibitors for their effects against RSV infection. Axl inhibition with kinase inhibitors, including BMS-777607, R428, and TP-0903, or Axl ablation resulted in a significant reduction of RSV infection in cell-based assays. In an animal model of pulmonary RSV infection, treatment with BMS-777607, R428, or TP-0903 ameliorated pulmonary pathology with a significant reduction of RSV titers in the lung tissues and, consequently, decreased the expression of proinflammatory genes. The host promotes ISG expression for the antiviral response and for viral clearance. We found that Axl inhibition led to more robust IFN-ß expression and antiviral gene induction. Thus, the results of this study imply that Axl kinase inhibitors may possess a broad spectrum of antiviral effects by promoting ISG expression.

Axl Mediates Resistance to Respiratory Syncytial Virus Infection Independent of Cell Attachment.

Respiratory syncytial virus (RSV) is a leading cause of severe lower respiratory tract infections in infants and young children. Axl, a TAM family receptor tyrosine kinase, has been demonstrated to be a receptor mediating enveloped virus infection. Here we show that Axl functions as a suppressor of antiviral response during RSV infection. Knockdown of Axl expression in human cells resulted in cell resistance to RSV infection, although the treatment did not significantly affect RSV binding or cell entry. Mice deficient in Axl showed resistance to RSV infection, including reduction in viral load and in pulmonary injury. Although T lymphocyte and macrophage infiltration was reduced, more IFN-γ-producing cells were present in BAL fluid in Axl-/- mice. Fewer alternatively activated alveolar macrophages were found in the lungs of Axl-/- mice. Axl-/- mouse embryonic fibroblasts and siRNA-treated human cells had more robust IFN-ß and IFN-stimulated gene induction of antiviral genes. Furthermore, reexpression of Axl using adenovirus-mediated Axl delivery repressed IFN-stimulated gene induction in Axl-null mouse embryonic fibroblasts by RSV infection. The results suggest that Axl, independent of being a virus entry receptor of RSV infection, negatively regulates IFN signaling to modulate host antiviral response against RSV infection.

Adenovirus is prevalent in juvenile polyps and correlates with low vitamin D receptor expression.

BACKGROUND: The objective of this study was to assess human adenovirus (HAdV) infection in juvenile polyps (JPs) and to preliminarily establish a correlation to vitamin D receptor (VDR) expression. METHODS: The study includes 76 patients of 5.2 ± 2.8 years old. Seventy-eight JP specimens and 24 parapolyp tissues from polypectomy were used. PCR was used to detect HAdV DNA and quantitative reverse transcription-PCR for viral and host gene expression. The PCR products were sequenced for virus typing. The correlation between VDR expression and HAdV infection was established using nonparametric Spearman's analysis. RESULTS: Seventy-four children (97.4%) had a single polyp and two had two polyps. The histopathological characteristics of the polyps were in line with JP. Thirty-three samples had HAdV DNA (43.4%), including 32 subgroup C and 1 subgroup B HAdV; no enteric HAdV was detected. HAdV messenger RNA was detected in 5 of the 33 samples (15.2%). The samples had increased interleukin-1ß (IL-1ß), IL-6, and calprotectin expression, and reduced E-cadherin and VDR expression. JP samples with low VDR expression were more prevalent of HAdV DNA (r = 1.261, 95% confidence interval, 1.017-1.563), while VDR expression positively correlated with E-cadherin and negatively with inflammation gene expression. CONCLUSIONS: HAdV latent infection was prevalent among JP tissues. The presence of HAdV correlated positively to low VDR expression. IMPACT: The HAdVs infect the upper airways and gastrointestinal system and is found to persist in lymphoid tissues. The prevalence of HAdV and the status of the infection is unknown. The study investigated the prevalence of HAdV from polypectomy specimens of JP patients and found that HAdV was prevalent and was in a persistent state. HAdV infection was more prevalent in samples with low VDR expression. Whether HAdV infection and reactivation is a contributing factor to JPs is unknown. Factors such as proinflammation and bacterial metabolites that are known to promote HAdV reactivation warrant further investigation.

Dynamic Changes in Lung Function and Imaging in Patients with COVID-19.

Purpose: To investigate the recovery of lung function and chest imaging in patients with COVID-19 three months after clinical cure and discharge and the correlation between them. Methods: This study collected 80 patients diagnosed with 2019-nCoV infection who were discharged from the Taizhou Public Health Medical Center in Zhejiang Province between January 31, 2020, and March 10, 2020. Lung function examinations and lung CT scans were performed at discharge and three months after discharge. The dynamic changes examined at discharge and three months after discharge were observed, and their correlation was analyzed. All data collection ended on June 25, 2020. Results: Among the 80 COVID-19 patients discharged from the hospital, the rate of abnormality indicated by lung CT images was 97.5%, mainly presenting as patchy shadows (95%), ground-glass shadows (75%), grid-like lesions, interlobular septal thickening or fiber strip shadows (30%), consolidation shadows, and nodules (10 cases each). At discharge, 72 patients (90%) had pulmonary dysfunction, 64 (80%) had restrictive ventilatory dysfunction, and 48 (60%) had small airway dysfunction. Three months after discharge, the rate of abnormality indicated by lung CT images was 12.5%. Eight cases (10%) showed residual patchy shadows, but the density was weak, and the scope was reduced. Two cases (2.5%) showed nodular shadows. Three months after discharge, 18 patients (22.5%) had residual restrictive ventilatory dysfunction, 28 patients (35%) had small airway dysfunction, and 32 patients (40%) had diffuse dysfunction. Moreover, patients with more severe chest imaging manifestations (bilateral lesions and ground-glass shadows combined with interstitial lesions) also had more obvious lung function impairment. Conclusion: Three months after being clinically cured, patients with COVID-19 had good chest imaging absorption and no residual fibrosis. Some patients had mild to moderate pulmonary dysfunction, mainly restricted ventilation dysfunction, small airway dysfunction, and diffuse dysfunction.

Histone Deacetylase Inhibitors Promote Latent Adenovirus Reactivation from Tonsillectomy Specimens.

Adenovirus (HAdV) infection is a common cause of illness among young children, immunocompromised patients, and transplant recipients. The majority of HAdV infections are self-limited, but recurring infection is frequently encountered in young children and may require hospitalization. In this study, we surveyed the presence of HAdV in tonsillectomy samples and investigated epigenetic conditions that contributed to HAdV reactivation. HAdV DNA was detected from 86.7% donors. The lymphocytes isolated from the samples failed to produce infectious HAdV after incubation, suggesting the viruses remained in a latent status. To determine whether epigenetic factors played a role in HAdV reactivation, isolated lymphocytes were treated with a small compound library. Viral DNA replication and infectious HAdV production were assayed by PCR and by a secondary infection assay. We identified several compounds, mainly pan- and selective histone deacetylase (HDAC) inhibitors, which showed activity to reactivate HAdV from latency. The viruses were isolated and were determined as species C HAdV. Using a model of HAdV lytic infection, we showed that the compounds promoted histone-3 acetylation and association with viral early gene promoters. In addition to demonstrate the palatine tonsils as a reservoir of latent HAdV, this study uncovers a critical role of histone acetylation in HAdV reactivation, linking HAdV latency to recurrent HAdV infection.IMPORTANCE Respiratory tract infection by adenoviruses is among the most common diseases in children, attributing to approximately 20% of hospitalizations of children with acute respiratory infection (ARI). Adenovirus transmits by direct contact, but recurrent infection is common. Ever since its isolation, adenovirus has been known to have the ability to establish persistent or latent infection. We found 87.7% tonsillectomy specimens contained detectable amounts of adenoviral DNA. Isolated lymphocytes did not produce infectious adenoviruses without stimulation. By screening an epigenetic informer compound library, we identified several histone deacetylase inhibitors that promoted adenovirus reactivation that was evidenced by increased viral DNA replication and production of infectious viruses. The human tonsils are covered with bacterial pathogens that may utilize pathogen-associated pattern molecules or metabolites to cause epigenetic activation and proinflammatory gene transcription, which may lead to viral reactivation from latency. The study shows that recurrent adenovirus infection could arise from reactivation of residing virus from previous infections.

Glycyrrhizic Acid Inhibits SARS-CoV-2 Infection by Blocking Spike Protein-Mediated Cell Attachment.

Glycyrrhizic acid (GA), also known as glycyrrhizin, is a triterpene glycoside isolated from plants of Glycyrrhiza species (licorice). GA possesses a wide range of pharmacological and antiviral activities against enveloped viruses including severe acute respiratory syndrome (SARS) virus. Since the S protein (S) mediates SARS coronavirus 2 (SARS-CoV-2) cell attachment and cell entry, we assayed the GA effect on SARS-CoV-2 infection using an S protein-pseudotyped lentivirus (Lenti-S). GA treatment dose-dependently blocked Lenti-S infection. We showed that incubation of Lenti-S virus, but not the host cells with GA prior to the infection, reduced Lenti-S infection, indicating that GA targeted the virus for infection. Surface plasmon resonance measurement showed that GA interacted with a recombinant S protein and blocked S protein binding to host cells. Autodocking analysis revealed that the S protein has several GA-binding pockets including one at the interaction interface to the receptor angiotensin-converting enzyme 2 (ACE2) and another at the inner side of the receptor-binding domain (RBD) which might impact the close-to-open conformation change of the S protein required for ACE2 interaction. In addition to identifying GA antiviral activity against SARS-CoV-2, the study linked GA antiviral activity to its effect on virus cell binding.

A positive feedback loop between ZNF205-AS1 and EGR4 promotes non-small cell lung cancer growth.

Accumulating evidences revealed that long noncoding RNAs (lncRNAs) are frequently implicated in non-small cell lung cancer (NSCLC). Herein, we reported the identification of a novel NSCLC-associated functional lncRNA ZNF205 antisense RNA 1 (ZNF205-AS1). ZNF205-AS1 was increased in NSCLC tissues and cell lines, and associated with poor prognosis of NSCLC patients. Bioinformatics prediction, combined with experimental verification revealed that early growth response 4 (EGR4) directly bound to ZNF205-AS1 promoter, increased the promoter activity of ZNF205-AS1, and activated ZNF205-AS1 transcription. Intriguingly, ZNF205-AS1 transcript directly interacted with EGR4 mRNA, increased EGR4 mRNA stability, and up-regulated EGR4 expression via RNA-RNA interaction. Thus, ZNF205-AS1 and EGR4 formed a positive feedback loop. Through regulating EGR4, ZNF205-AS1 activated its own promoter activity. EGR4 was also increased in NSCLC and the expression of ZNF205-AS1 was significantly positively correlated with EGR4 in NSCLC tissues. Gain-of-function and loss-of-function assays demonstrated that both ZNF205-AS1 and EGR4 promoted NSCLC cell growth in vitro and NSCLC tumour growth in vivo. Concurrently depleting ZNF205-AS1 and EGR4 more significantly repressed NSCLC tumour growth in vivo. Collectively, our study demonstrated that the positive feedback loop between ZNF205-AS1 and EGR4 promotes NSCLC growth, and implied that targeting this feedback loop may be promising therapeutic strategy for NSCLC.

Single strand transposition at the host replication fork.

Members of the IS200/IS605 insertion sequence family differ fundamentally from classical IS essentially by their specific single-strand (ss) transposition mechanism, orchestrated by the Y1 transposase, TnpA, a small HuH enzyme which recognizes and processes ss DNA substrates. Transposition occurs by the 'peel and paste' pathway composed of two steps: precise excision of the top strand as a circular ss DNA intermediate; and subsequent integration into a specific ssDNA target. Transposition of family members was experimentally shown or suggested by in silico high-throughput analysis to be intimately coupled to the lagging strand template of the replication fork. In this study, we investigated factors involved in replication fork targeting and analysed DNA-binding properties of the transposase which can assist localization of ss DNA substrates on the replication fork. We showed that TnpA interacts with the ß sliding clamp, DnaN and recognizes DNA which mimics replication fork structures. We also showed that dsDNA can facilitate TnpA targeting ssDNA substrates. We analysed the effect of Ssb and RecA proteins on TnpA activity in vitro and showed that while RecA does not show a notable effect, Ssb inhibits integration. Finally we discuss the way(s) in which integration may be directed into ssDNA at the replication fork.

A Model for Transposition of the Colistin Resistance Gene mcr-1 by ISApl1.

Analysis of mcr-1-containing sequences identified a common ∼2,607-bp DNA segment that in many cases is flanked on one or both ends by ISApl1 We present evidence that mcr-1 is mobilized by an ISApl1 composite transposon which has, in some cases, subsequently lost one or both copies of ISApl1 We also show that mcr-1 can be mobilized in some circumstances by a single upstream copy of ISApl1 in conjunction with the remnants of a downstream ISApl1.

IS200/IS605 family single-strand transposition: mechanism of IS608 strand transfer.

Transposase, TnpA, of the IS200/IS605 family member IS608, catalyses single-strand DNA transposition and is dimeric with hybrid catalytic sites composed of an HUH motif from one monomer and a catalytic Y127 present in an α-helix (αD) from the other (trans configuration). αD is attached to the main body by a flexible loop. Although the reactions leading to excision of a transposition intermediate are well characterized, little is known about the dynamic behaviour of the transpososome that drives this process. We provide evidence strongly supporting a strand transfer model involving rotation of both αD helices from the trans to the cis configuration (HUH and Y residues from the same monomer). Studies with TnpA heterodimers suggest that TnpA cleaves DNA in the trans configuration, and that the catalytic tyrosines linked to the 5'-phosphates exchange positions to allow rejoining of the cleaved strands (strand transfer) in the cis configuration. They further imply that, after excision of the transposon junction, TnpA should be reset to a trans configuration before the cleavage required for integration. Analysis also suggests that this mechanism is conserved among members of the IS200/IS605 family.

Molecular basis for the transcriptional regulation of an epoxide-based virulence circuit in Pseudomonas aeruginosa.

The opportunistic pathogen Pseudomonas aeruginosa infects cystic fibrosis (CF) patient airways and produces a virulence factor Cif that is associated with worse outcomes. Cif is an epoxide hydrolase that reduces cell-surface abundance of the cystic fibrosis transmembrane conductance regulator (CFTR) and sabotages pro-resolving signals. Its expression is regulated by a divergently transcribed TetR family transcriptional repressor. CifR represents the first reported epoxide-sensing bacterial transcriptional regulator, but neither its interaction with cognate operator sequences nor the mechanism of activation has been investigated. Using biochemical and structural approaches, we uncovered the molecular mechanisms controlling this complex virulence operon. We present here the first molecular structures of CifR alone and in complex with operator DNA, resolved in a single crystal lattice. Significant conformational changes between these two structures suggest how CifR regulates the expression of the virulence gene cif. Interactions between the N-terminal extension of CifR with the DNA minor groove of the operator play a significant role in the operator recognition of CifR. We also determined that cysteine residue Cys107 is critical for epoxide sensing and DNA release. These results offer new insights into the stereochemical regulation of an epoxide-based virulence circuit in a critically important clinical pathogen.

An engineered TNFR1-selective human lymphotoxin-alpha mutant delivered by an oncolytic adenovirus for tumor immunotherapy.

Lymphotoxin α (LTα) is a soluble factor produced by activated lymphocytes which is cytotoxic to tumor cells. Although a promising candidate in cancer therapy, the application of recombinant LTα has been limited by its instability and toxicity by systemic administration. Secreted LTα interacts with several distinct receptors for its biological activities. Here, we report a TNFR1-selective human LTα mutant (LTα Q107E) with potent antitumor activity. Recombinant LTα Q107E with N-terminal 23 and 27 aa deletion (named LTα Q1 and Q2, respectively) showed selectivity to TNFR1 in both binding and NF-κB pathway activation assays. To test the therapeutic potential, we constructed an oncolytic adenovirus (oAd) harboring LTα Q107E Q2 mutant (named oAdQ2) and assessed the antitumor effect in mouse xenograft models. Intratumoral delivery of oAdQ2 inhibited tumor growth. In addition, oAdQ2 treatment enhanced T cell and IFNγ-positive CD8 T lymphocyte infiltration in a human PBMC reconstituted-SCID mouse xenograft model. This study provides evidence that reengineering of bioactive cytokines with tissue or cell specific properties may potentiate their therapeutic potential of cytokines with multiple receptors.

Reconstitution of a functional IS608 single-strand transpososome: role of non-canonical base pairing.

Single-stranded (ss) transposition, a recently identified mechanism adopted by members of the widespread IS200/IS605 family of insertion sequences (IS), is catalysed by the transposase, TnpA. The transposase of IS608, recognizes subterminal imperfect palindromes (IP) at both IS ends and cleaves at sites located at some distance. The cleavage sites, C, are not recognized directly by the protein but by short sequences 5' to the foot of each IP, guide (G) sequences, using a network of canonical ('Watson-Crick') base interactions. In addition a set of non-canonical base interactions similar to those found in RNA structures are also involved. We have reconstituted a biologically relevant complex, the transpososome, including both left and right ends and TnpA, which catalyses excision of a ss DNA circle intermediate. We provide a detailed picture of the way in which the IS608 transpososome is assembled and demonstrate that both C and G sequences are essential for forming a robust transpososome detectable by EMSA. We also address several questions central to the organization and function of the ss transpososome and demonstrate the essential role of non-canonical base interactions in the IS608 ends for its stability by using point mutations which destroy individual non-canonical base interactions.

Case Report: Keratoacanthoma and type I diabetes secondary to treatment with PM8001, a bifunctional fusion protein targeting TGF-ß and PD-L1.

Immune-related adverse reactions primarily involve the skin and the endocrine, digestive, and respiratory systems. In the endocrine system, these adverse effects mainly include hypophysitis, thyroiditis, hypoadrenalism, and rarely, diabetes mellitus. The most common symptoms in the skin are pruritus, rash, and infrequently, eruptive keratoacanthoma. Here, we report a case of a 67-year-old woman who developed eruptive keratoacanthoma of the skin 6 weeks after beginning treatment with a bispecific antibody (PM8001), targeting both programmed cell death receptor 1 and transforming growth factor ß, as well as type I diabetes mellitus-induced ketoacidosis after 13 weeks. The type I diabetes appeared to stabilize after insulin treatment, and the keratoacanthoma gradually resolved after drug discontinuation. This case report describes a case of the effects of PM8001 immunotherapy on the endocrine glands and skin, together with a review of the relevant literature, and summarizes the different clinical characteristics of rare immune-related adverse events resulting from PM8001 immunotherapy to provide a reference for their early detection, diagnosis, and treatment.

Efficacy and safety of re-challenging 160 mg furmonertinib for advanced NSCLC after resistance to third-generation EGFR-TKIs targeted agents: A real-world study.

BACKGROUND: Third-generation epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI) show good selectivity for classical EGFR mutated and EGFR T790M mutated non-small cell lung cancer (NSCLC). However, resistance inevitably occurs to third-generation EGFR-TKI. This study describes the real-world characteristics, efficacy, and safety of treating post-progression NSCLC with 160 mg of furmonertinib (in combination with or without anti-angiogenic agents and chemotherapy) with third-generation EGFR-TKIs. METHODS: EGFR-mutated NSCLC patients with intracranial progression pattern cohort (IP cohort) or extracranial progression pattern cohort (EP cohort) were retrospectively analyzed following progression to third-generation EGFR-TKIs receiving furmonertinib 160 mg daily as second-line or later treatment in combination with or without anti-angiogenic agents and chemotherapy. RESULTS: Thirty-nine patients were included and categorized into two groups according to the progression pattern. Then, 22 patients in the IP cohort and 17 patients in the EP cohort, most of whom were in poor physical condition, were included and 84.6% had central nervous system metastases. In the IP cohort, the median PFS was 5.5 months (95% CI 4.67-8.72), and the median OS was 9.8 months (95% CI 7.25-11.20) for single-agent furmonertinib or combination therapy. In the EP cohort, the median PFS was 3.2 months (95% CI 2.18-4.70), and the median OS was 6.7 months (95% CI 4.99-8.75). Univariate analysis showed the association between the presence of a prior T790M mutation and a history of combined radiotherapy with longer PFS with furmonertinib (p = 0.048, p = 0.004). Overall, adverse events (AEs) of any grade occurred in 84.6% of patients (33/39), with the majority having grade 2 or lower AEs. CONCLUSION: Furmonertinib 160 mg is an optional regimen for patients with advanced NSCLC who develop resistance after treatment with third-generation EGFR-TKIs, especially those developing resistance due to the progression of intracranial lesions, with good efficacy and an acceptable safety profile that warrants further exploration.

Response from Varani et al. to "Comment on 'the IS6 family, a clinically important group of insertion sequences including IS26' by Ruth M. Hall".

The IS6 family of insertion sequences is a large but coherent group which was originally named to avoid confusion between a number of identical or nearly identical IS that were identified at about the same time and given different names (IS15D, IS26, IS46, IS140, IS160, IS176). The underlying common mechanistic feature of all IS6 family members which have been investigated is that they appear to transpose by replicative transposition and form pseudo compound transposons with the flanking IS in direct repeat and in which associated genes are simply transferred to the target replicon and lost from the donor.In the accompanying letter Hall raises a number of very serious and wide-ranging criticisms of our recent review article concerning the IS6 family of insertion sequences. She clearly feels that we have undervalued her work and that we question or ignore certain of her in vivo results. This impression is almost certainly the result of the standard of proof we generally apply to mechanistic aspects of transposition where we think it important to identify transposition intermediates including the types of synaptic, strand cleavage and strand transfer complexes involved.

Current status and challenges of immunotherapy in ALK rearranged NSCLC.

Rearrangements of the anaplastic lymphoma kinase (ALK) gene account for 5-6% in non-small cell lung cancer (NSCLC). ALK rearranged NSCLC is sensitive to ALK tyrosine kinase inhibitors (TKIs) but prone to drug resistance. Meanwhile, ALK rearranged NSCLC has poor response to single immunotherapy. Here we mainly describe the immune escape mechanisms of ALK mutated NSCLC and the role of related biomarkers. Additionally, we collate and evaluate preclinical and clinical studies of novel immune combination regimens, and describe the prospects and perspectives for the in vivo application of novel immune technologies in patients with ALK rearranged NSCLC.