Optimizing phage-based mutant recovery and minimizing heat effect in the construction of transposon libraries in Staphylococcus aureus.

Staphylococcus aureus (S. aureus), particularly Methicillin-resistant S. aureus (MRSA), poses a significant global public health threat, necessitating advanced methodologies to enhance our understanding of this organism at the omics levels. This study introduces a refined protocol for constructing and curing high-density transposon mutant (tn-mutant) libraries in S. aureus, addressing the challenges associated with low transductant yields, and the complex genetic manipulation mechanism in Gram-positive bacteria. Our methodology employs a Himar1 transposon based on a two-plasmid system, leveraging Himar1's high insertional efficiency in AT-rich organisms. Enhanced transduction efficiency was achieved through chloramphenicol pre-treatment and the use of modified enriched media. Complementing this, an optimized plasmid curing procedure ensured a representative and stable tn-mutant library. The protocol was successfully applied to multiple S. aureus strains, demonstrating an increase in mutant recovery and reduced post-curing impact. The method offers a robust approach for Transposon Insertion Sequencing (TIS) applications in S. aureus, enabling deeper insights into survival, resistance, and pathogenicity mechanisms. This protocol holds a significant potential for accelerating the construction of tn-mutant libraries in various S. aureus strains.

High fidelity DNA ligation prevents single base insertions in the yeast genome.

Finalization of eukaryotic nuclear DNA replication relies on DNA ligase 1 (LIG1) to seal DNA nicks generated during Okazaki Fragment Maturation (OFM). Using a mutational reporter in Saccharomyces cerevisiae, we previously showed that mutation of the high-fidelity magnesium binding site of LIG1Cdc9 strongly increases the rate of single-base insertions. Here we show that this rate is increased across the nuclear genome, that it is synergistically increased by concomitant loss of DNA mismatch repair (MMR), and that the additions occur in highly specific sequence contexts. These discoveries are all consistent with incorporation of an extra base into the nascent lagging DNA strand that can be corrected by MMR following mutagenic ligation by the Cdc9-EEAA variant. There is a strong preference for insertion of either dGTP or dTTP into 3-5 base pair mononucleotide sequences with stringent flanking nucleotide requirements. The results reveal unique LIG1Cdc9-dependent mutational motifs where high fidelity DNA ligation of a subset of OFs is critical for preventing mutagenesis across the genome.

Experimental considerations for precise RNA-mediated insertion of transgenes.

Precise RNA-mediated insertion of transgenes (PRINT) is a pioneering method for site-specific, safe-harbor transgene supplementation of the human genome that harnesses a eukaryotic retroelement protein and relies solely on the delivery of RNA. Here we outline important considerations in the design of the two required RNAs, details for the production and transfection of these RNAs to cells, and read-outs for successful transgene addition. Throughout, tips and key concepts are laid out to enable general use of this method.

Isolation and characterization of a novel S1-gene insertion porcine epidemic diarrhea virus with low pathogenicity in newborn piglets.

Porcine epidemic diarrhea virus (PEDV) causes diarrhea and vomiting in piglets, leading to a mortality rate of 100%. Due to the high frequency of mutation, it is important to monitor the evolution of PEDV and develop potential vaccine candidates. In this study, two PEDV strains (ZJ2022 and ZQ2022) were identified by PCR. These strains were subsequently isolated, and their genome sequences, growth characteristics, and pathogenicity were compared. Phylogenetic and recombination analyses revealed that both strains belonged to GIIa-subgroup, and ZQ2022 was identified as a recombinant strain derived from ZJ2022. Further sequence analysis showed that the ZJ2022 strain had a modified top region of the S1 protein due to a three amino acid insertion (T380_Y380insGGE) in the S1 gene. According to the virus growth curve, ZJ2022 exhibited better cellular adaptation than ZQ2022, with higher viral titers from 8 hpi to 24 hpi. Additionally, ZQ2022 exhibited a high level of pathogenicity, causing severe diarrhea in piglets at 36 hpi and a 100% mortality rate by 96 hpi. In contrast, ZJ2022 showed lower pathogenicity, inducing severe diarrhea in piglets at 60 hpi, with a mortality rate of 60% at 96 hpi and 100% at 120 hpi. In summary, our findings provided evidence of the undergoing mutations in Chinese PEDV strains. Furthermore, the S gene insertion strain ZJ2022 exhibited strong cellular adaptability and low pathogenicity, making it a potential candidate strain for vaccine development.

Identifying transgene insertions in Caenorhabditis elegans genomes with Oxford Nanopore sequencing.

Genetically modified organisms are commonly used in disease research and agriculture but the precise genomic alterations underlying transgenic mutations are often unknown. The position and characteristics of transgenes, including the number of independent insertions, influences the expression of both transgenic and wild-type sequences. We used long-read, Oxford Nanopore Technologies (ONT) to sequence and assemble two transgenic strains of Caenorhabditis elegans commonly used in the research of neurodegenerative diseases: BY250 (pPdat-1::GFP) and UA44 (GFP and human α-synuclein), a model for Parkinson's research. After scaffolding to the reference, the final assembled sequences were ∼102 Mb with N50s of 17.9 Mb and 18.0 Mb, respectively, and L90s of six contiguous sequences, representing chromosome-level assemblies. Each of the assembled sequences contained more than 99.2% of the Nematoda BUSCO genes found in the C. elegans reference and 99.5% of the annotated C. elegans reference protein-coding genes. We identified the locations of the transgene insertions and confirmed that all transgene sequences were inserted in intergenic regions, leaving the organismal gene content intact. The transgenic C. elegans genomes presented here will be a valuable resource for Parkinson's research as well as other neurodegenerative diseases. Our work demonstrates that long-read sequencing is a fast, cost-effective way to assemble genome sequences and characterize mutant lines and strains.

Natural Transposable Element Insertions Contribute to Host Fitness in Model Yeasts.

Transposable elements (TEs) are ubiquitous in the eukaryote genomes, but their evolutionary and functional significance remains largely obscure and contentious. Here, we explore the evolution and functional impact of TEs in two model unicellular eukaryotes, the fission yeast Schizosaccharomyces pombe and the budding yeast Saccharomyces cerevisiae, which diverged around 330 to 420 million years ago. We analyze the distribution of LTR retrotransposons (LTR-RTs, the only TE order identified in both species) and their solo-LTR derivatives in 35 strains of S. pombe and 128 strains of S. cerevisiae. We find that natural LTR-RT and solo-LTR insertions exhibit high presence-absence polymorphism among individuals in both species. Population genetics analyses show that solo-LTR insertions experienced functional constraints similar to synonymous sites of host genes in both species, indicating a majority of solo-LTR insertions might have evolved in a neutral manner. When knocking out nine representative solo-LTR insertions separately in the S. pombe strain 972h- and 12 representative solo-LTR insertions separately in the S. cerevisiae strain S288C, we find that one solo-LTR insertion in S. pombe has a significant effect on the fitness and transcriptome of its host. Together, our findings indicate that a fraction of natural TE insertions likely shape their host transcriptomes and thereby contribute to their host fitness, with implications for understanding the functional significance of TEs in eukaryotes.

[EGFR Exon 20 Insertion Mutation: Research Status and New Treatment Strategies].

In non-small cell lung cancer (NSCLC), as an improtant oncogenic driver gene, epidermal growth factor receptor exon 20 insertion (EGFR ex20ins) has a unique protein structure and is primarily drug-resistant to traditional EGFR-tyrosine kinase inhibitors (EGFR-TKIs). In recent years, exploration of targeted therapy for EGFR ex20ins has never stopped. Firstly Mobocertinib and Amivantamab obtained approval from U.S. Food and Drug Administration (FDA) for EGFR ex20ins mutant NSCLC patients, then other drugs, such as Sunvozertinib, made breakthroughs and combination therapies also obtained gains. Multi-pronged measures are hopeful to overcome EGFR ex20ins drug resistance. As mentioned above, it's definitely important to gain deeper understanding of molecular mechanism of EGFR ex20ins and assess effect and difference between novel drugs. This review is devoted to make a summary about newest achievement so to provide valuable reference about precise therapy for patients with EGFR ex20ins.
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Alu insertion-mediated dsRNA structure formation with pre-existing Alu elements as a disease-causing mechanism.

We previously identified a homozygous Alu insertion variant (Alu_Ins) in the 3'-untranslated region (3'-UTR) of SPINK1 as the cause of severe infantile isolated exocrine pancreatic insufficiency. Although we established that Alu_Ins leads to the complete loss of SPINK1 mRNA expression, the precise mechanisms remained elusive. Here, we aimed to elucidate these mechanisms through a hypothesis-driven approach. Initially, we speculated that, owing to its particular location, Alu_Ins could independently disrupt mRNA 3' end formation and/or affect other post-transcriptional processes such as nuclear export and translation. However, employing a 3'-UTR luciferase reporter assay, Alu_Ins was found to result in only an ∼50% reduction in luciferase activity compared to wild type, which is insufficient to account for the severe pancreatic deficiency in the Alu_Ins homozygote. We then postulated that double-stranded RNA (dsRNA) structures formed between Alu elements, an upstream mechanism regulating gene expression, might be responsible. Using RepeatMasker, we identified two Alu elements within SPINK1's third intron, both oriented oppositely to Alu_Ins. Through RNAfold predictions and full-length gene expression assays, we investigated orientation-dependent interactions between these Alu repeats. We provide compelling evidence to link the detrimental effect of Alu_Ins to extensive dsRNA structures formed between Alu_Ins and pre-existing intronic Alu sequences, including the restoration of SPINK1 mRNA expression by aligning all three Alu elements in the same orientation. Given the widespread presence of Alu elements in the human genome and the potential for new Alu insertions at almost any locus, our findings have important implications for detecting and interpreting Alu insertions in disease genes.

Current clinical practice and physicians' insights on Chinese patients with advanced non-small cell lung cancer habouring epidermal growth factor receptor 20 insertion mutation.

BACKGROUND: The present study aimed to investigate physicians' perspectives on the diagnosis and treatment decisions for patients with non-small cell lung cancer (NSCLC) harbouring epidermal growth factor receptor (EGFR) exon 20 insertion (exon20ins) mutations in a real-world setting in China using an online questionnaire. METHODS: This study was performed via the CAPTRA-Lung collaboration between December 9, 2022 and March 6, 2023. The questionnaire was distributed digitally to physicians around China and was comprised of three sections: basic characteristics of surveyed physicians, diagnosis and treatment status of NSCLC patients with the EGFR exon20ins-mutation, and physicians' perspectives on treatment options. Physicians who treat more than 10 patients with advanced NSCLC every month and who have treated patients with advanced EGFR exon20ins-mutant NSCLC in the past six months were involved in this study. RESULTS: A total of 53,729 questionnaires were distributed and 390 valid ones were collected. The EGFR mutation test was performed in 80.9% and 59.9% of patients receiving first-line or second-line therapy and beyond (hereinafter "second-line")therapy, respectively. In terms of treatment options, chemotherapy plus antiangiogenic therapy was the most common treatment option (30.0% of patients in first-line settings; 25.0% of patients in second-line settings), and a certain proportion of patients received novel EGFR exon20ins-targeted agents (including tyrosine kinase inhibitors [TKIs] and bispecific antibodies) in first- or second-line settings, which accounted for 11.9% and 15.7% of all treated patients, respectively. Additionally, physicians reported the highest satisfaction score for the efficacy and safety of targeted agents. Most physicians believed that EGFR exon20ins-targeted TKIs represented the most promising treatment option (80.2% in first-line treatment and 73.3% in second-line treatment). Among several novel agents under study, sunvozertinib has received the highest recognition for efficacy and safety. CONCLUSIONS: This study investigated the current diagnosis and treatment status and physicians' perspective, of patients with EGFR exon20ins-mutant NSCLC. The results highlight significant unmet clinical needs in this subgroup of patients. EGFR exon20ins-targeted TKIs were recognized as the most promising treatment regimen and may benefit more patients considering their awareness and acceptance of targeted therapy.

Screening of pathogenicity-deficient Penicillium italicum mutants established by Agrobacterium tumefaciens-mediated transformation.

Blue mold, caused by Penicillium italicum, is one of the main postharvest diseases of citrus fruits during storage and marketing. The pathogenic mechanism remains largely unclear. To explore the potential pathogenesis-related genes of this pathogen, a T-DNA insertion library of P. italicum PI5 was established via Agrobacterium tumefaciens-mediated transformation (ATMT). The system yielded 200-250 transformants per million conidia, and the transformants were genetically stable after five generations of successive subcultures on hygromycin-free media. 2700 transformants were obtained to generate a T-DNA insertion library of P. italicum. Only a few of the 200 randomly selected mutants exhibited significantly weakened virulence on citrus fruits, with two mutants displaying attenuated sporulation. The T-DNA in the two mutants existed as a single copy. Moreover, the mutant genes PiBla (PITC_048370) and PiFTF1 (PITC_077280) identified may be involved in conidia production by regulating expressions of the key regulatory components for conidiogenesis. These results demonstrated that the ATMT system is useful to obtain mutants of P. italicum for further investigation of the molecular mechanisms of pathogenicity and the obtained two pathogenesis-related genes might be novel loci associated with pathogenesis and conidia production.

Transposon mutagenesis screen in Klebsiella pneumoniae identifies genetic determinants required for growth in human urine and serum.

Klebsiella pneumoniae is a global public health concern due to the rising myriad of hypervirulent and multidrug-resistant clones both alarmingly associated with high mortality. The molecular mechanisms underpinning these recalcitrant K. pneumoniae infection, and how virulence is coupled with the emergence of lineages resistant to nearly all present-day clinically important antimicrobials, are unclear. In this study, we performed a genome-wide screen in K. pneumoniae ECL8, a member of the endemic K2-ST375 pathotype most often reported in Asia, to define genes essential for growth in a nutrient-rich laboratory medium (Luria-Bertani [LB] medium), human urine, and serum. Through transposon directed insertion-site sequencing (TraDIS), a total of 427 genes were identified as essential for growth on LB agar, whereas transposon insertions in 11 and 144 genes decreased fitness for growth in either urine or serum, respectively. These studies not only provide further knowledge on the genetics of this pathogen but also provide a strong impetus for discovering new antimicrobial targets to improve current therapeutic options for K. pneumoniae infections.

Tumor immune microenvironment of NSCLC with EGFR exon 20 insertions may predict efficacy of first-line ICI-combined regimen.

OBJECTIVES: Non-small cell lung cancer (NSCLC) patients with exon 20 insertion mutations (ex20ins) of the epidermal growth factor receptor (EGFR) were resistant to monotherapy of immune checkpoint inhibitor (ICI). However, recent reports have shown that the combination of ICI and chemotherapy (ICI-combined regimen) exhibited certain efficacy for NSCLC with EGFR ex20ins. The mechanisms behind this phenomenon have not been thoroughly clarified. Hence, we conducted this study tofind correlations between the tumor immune microenvironment of EGFR ex20ins and the efficacy of ICI-combined regimen. METHODS: We performed single-cell transcriptome sequencing and multiplex immunofluorescence staining (mIF) to investigate the immune microenvironment of NSCLC patients with EGFR ex20ins, L858R, and EGFR wild-type. We analyzed 15 treatment-naïve NSCLC samples utilizing single-cell RNA sequencing (scRNA-seq). Another 30 cases of EGFR L858R and 4 cases of wild-type were recruited to compare the immune microenvironment with that of EGFR ex20ins (28 cases) by mIF. RESULTS: We observed that cell components, function and interactions varied between EGFR ex20ins, L858R, and wild-type NSCLC.We discovered similar T cell and CD8+ T cell distributions among groups but found noninferior or even better T cell activation in ex20ins patients. Infiltrating CD8+ FOXP3- T cells were significantly lower in the tumor region of EGFR ex20ins compared to wild-type. T cells from the ex20ins group had a greater tendency to promote cancer cell inflammation and epithelial-mesenchymal transition (EMT) compared to wild-type group. For macrophages, there were more M2-like macrophages in ex20ins patients. M1-like macrophages in ex20ins group produced fewer antitumor cytokines than in other groups. CONCLUSIONS: The immune microenvironment of EGFR ex20ins is more suppressive than that of L858R and wild-type, suggesting that ICI monotherapy may not be sufficient for these patients. ICI-combined regimen might be a treatment option for EGFR ex20ins due to tumor-promoting inflammation and noninferior T cell functions in the immune microenvironment.

Protocol for generation of CRISPR-Cas9-mediated specific genomic insertion of P2A-Gal4 to reveal endogenous gene expression in Drosophila.

Generating a transgene with a reporter inserted into the genome helps us study endogenous gene expression patterns in model organisms. Here, using Drosophila melanogaster, we present a protocol for generating a P2A-Gal4 insertion through CRISPR-Cas9-mediated homology recombination. We describe the design strategy, steps for constructing the injection plasmids, and the fly-cross scheme for screening the transformants from the G0 generation. This protocol can also be applied to introduce mutations or various genetic tools into the fly genome. For complete details on the use and execution of this protocol, please refer to Li et al.1.

A phase 2 study of mobocertinib as first-line treatment in Japanese patients with non-small cell lung cancer harboring EGFR exon 20 insertion mutations.

BACKGROUND: Mobocertinib is a novel, synthetic, orally administered tyrosine kinase inhibitor that inhibits many activated forms of epidermal growth factor receptor (EGFR), including those containing exon 20 insertion (ex20ins) mutations. This study aimed to assess the efficacy of mobocertinib in Japanese patients with locally advanced or metastatic non-small cell lung cancer (NSCLC) harboring EGFR ex20ins mutations. METHODS: This was a phase 2, open-label study. Patients with NSCLC harboring EGFR ex20ins mutations who had not had previous systemic treatment received mobocertinib 160 mg once daily. The primary endpoint was the confirmed objective response rate. A planned interim analysis was completed for the first 14 patients with a centrally confirmed EGFR ex20ins mutation, with enrollment stopped if the number of patients with an objective response was five or fewer. RESULTS: In total, 33 patients were enrolled into the study (63.6% women; median age: 66 years). At the interim analysis, the objective response rate evaluated by a central independent review committee was 28.6% (4/14, 90% confidence interval: 10.4-54.0); therefore, enrollment was stopped for futility. In the full analysis set, the objective response rate was 18.2% (6/33, 95% confidence interval: 7.0-35.5); of the six responders, one patient (3.0%) had a complete response and five patients (15.2%) had partial responses. The most common treatment-related adverse events were diarrhea, paronychia, stomatitis, and nausea. CONCLUSION: Although study enrollment was terminated early owing to futility, our results showed modest activity of mobocertinib in Japanese patients with NSCLC with EGFR ex20ins mutations with no additional safety concerns.

Molecular heterogeneity and treatment outcome of EGFR exon 20 insertion mutations in Chinese patients with advanced non-small cell lung cancer: insights from a large-scale real-world study.

INTRODUCTION: This retrospective study aimed to investigate treatment patterns and outcomes in patients with NSCLC harboring EGFR20ins in China. EGFR20ins mutations are associated with poor responses to EGFR-TKIs, and limited real-world data exist regarding the efficacy of various treatment modalities. METHODS: In this retrospective, single-center study, treatment outcomes, including PFS and ORR, were evaluated for different treatment regimens based on imaging assessments. The impact of mutation heterogeneity on treatment efficacy was also explored. RESULTS: Data from 302 patients diagnosed with NSCLC with EGFR20ins were analyzed. EGFR-TKI monotherapy demonstrated suboptimal PFS compared to platinum-based chemotherapy in the first-line setting (3.00 m vs. 6.83 m, HR = 3.674, 95%CI = 2.48-5.44, p < 0.001). Platinum plus pemetrexed plus bevacizumab combination therapy showed improved PFS and ORR compared to platinum plus pemetrexed (7.50m vs. 5.43 m, HR = 0.593, 95%CI = 0.383-0.918, p = 0.019). In later-line treatments, monotherapy with EGFR-TKIs or ICIs exhibited suboptimal efficacy. The specific EGFR20ins subtype, A763_Y764insFQEA, showed favorable responses to EGFR-TKIs in real-world settings. CONCLUSIONS: This large-scale real-world study provides valuable insights into the treatment patterns and outcomes of NSCLC patients with EGFR20ins mutations in China. These findings contribute to the understanding of EGFR20ins treatment and provide real-world benchmark for future clinical trials and drug development.

[Chinese Expert Consensus on the Standardized Diagnosis and Treatment of 
Non­small Cell Lung Cancer with EGFR Exon 20 Insertion Mutations (2024 Edition)].

The standard clinical practice of managing the non-small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) exon 20 insertion (ex20ins) mutations was elaborated in Chinese expert consensus on non­small cell lung cancer with EGFR exon 20 insertion mutations (2023 edition), and this rare subset has gradually attracted attention recently. With the deepening of treatment area exploration and the approval of new targeted drugs, there are more options for the diagnosis and treatment of EGFR ex20ins positive NSCLC patients. Therefore, based on the previous version of consensus, the expert panel has updated this consensus on the standardized clinical diagnosis and treatment of EGFR ex20ins mutation NSCLC through reference to literature and clinical data, and combined with the experts' own clinical experience. The updated recommendations includes disease congnition, testing methods, therapy and recent relevant clinical trials for NSCLC patients with EGFR ex20ins mutation, in order to provide better medication reference for clinical physicians.
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A phase II study of osimertinib in patients with NSCLC harboring EGFR exon 20 insertion: A multicenter trial of the Korean Cancer Study Group (LU17-19).

BACKGROUND: Epidermal growth factor receptor (EGFR) exon 20 insertions account for up to 10% of all EGFR mutations. Clinical outcomes in patients receiving approved EGFR exon 20 insertion-specific inhibitors have been variable. Although osimertinib has demonstrated antitumor activity in clinical trials, its clinical efficacy and translational potential remain to be determined in non-small cell lung carcinoma (NSCLC) with EGFR exon 20 insertion. METHODS: In this multicenter phase II study, patients with advanced NSCLC harboring EGFR exon 20 insertions for whom the standard chemotherapy failed received 80 mg osimertinib once daily. The primary endpoint was the investigator-assessed objective response rate (ORR) as defined by Response Evaluation Criteria in Solid Tumors version 1.1. The secondary endpoints were progression-free survival (PFS), overall survival (OS), and safety profile. RESULTS: Among 15 patients enrolled at stage 1, the best response was most commonly disease stabilization (73.3 %), which did not meet the stage 1 threshold (objective response ≥ 2/15). As of data cutoff, two patients remained on the treatment. The median PFS and OS were 3.8 (95 % confidence interval [CI] = 1.7-5.5) months and 6.5 (95 % CI = 3.9-not reached) months, respectively. Adverse events (≥grade 3) were anemia, hypercalcemia, and pneumonia (13.3 % each), and asthenia, femur fracture, increased alkaline phosphate, hyperkalemia, bone pain, and azotemia (6.7 % each). Pre-existing EGFR C797S mutation detected in plasma limited the efficacy of osimertinib. CONCLUSION: Osimertinib at 80 mg once daily had limited efficacy and mostly showed disease stabilization with an acceptable safety profile in advanced NSCLC harboring EGFR exon 20 insertions. CLINICALTRIALS: govIdentifier: NCT03414814.

Mapping of long interspersed element-1 (L1) insertions by TIPseq provides information about sub chromosomal genetic variation in human embryos.

PURPOSE: Retrotransposons play important roles during early development when they are transiently de-repressed during epigenetic reprogramming. Long interspersed element-1 (L1), the only autonomous retrotransposon in humans, comprises 17% of the human genome. We applied the Single Cell Transposon Insertion Profiling by Sequencing (scTIPseq) to characterize and map L1 insertions in human embryos. METHODS: Sixteen cryopreserved, genetically tested, human blastocysts, were accessed from consenting couples undergoing IVF at NYU Langone Fertility Center. Additionally, four trios (father, mother, and embryos) were also evaluated. scTIPseq was applied to map L1 insertions in all samples, using L1 locations reported in the 1000 Genomes as controls. RESULTS: Twenty-nine unknown and unique insertions were observed in the sixteen embryos. Most were intergenic; no insertions were located in exons or immediately upstream of genes. The location or number of unknown insertions did not differ between euploid and aneuploid embryos, suggesting they are not merely markers of aneuploidy. Rather, scTIPseq provides novel information about sub-chromosomal structural variation in human embryos. Trio analyses showed a parental origin of all L1 insertions in embryos. CONCLUSION: Several studies have measured L1 expression at different stages of development in mice, but this study for the first time reports unknown insertions in human embryos that were inherited from one parent, confirming no de novo L1 insertions occurred in parental germline or during embryogenesis. Since one-third of euploid embryo transfers fail, future studies would be useful for understanding whether these sub-chromosomal genetic variants or de novo L1 insertions affect embryo developmental potential.

Metabolism and Excretion of [14C]Mobocertinib, a Selective Covalent Inhibitor of Epidermal Growth Factor Receptor (EGFR) Exon 20 Insertion Mutations, in Healthy Male Subjects.

Mobocertinib (formerly known as TAK-788) is a targeted covalent tyrosine kinase inhibitor of epidermal growth factor receptor with exon 20 insertion mutations. This article describes the metabolism and excretion of mobocertinib in healthy male subjects after a single oral administration of [14C]mobocertinib. Mobocertinib-related materials were highly covalently bound to plasma proteins such as human serum albumin. The mean extraction recovery of total radioactivity was only 3.9% for six individual Hamilton pooled plasma samples. After extraction, mobocertinib was the most abundant component accounting for 7.7% of total extracted circulating radioactivity (TECRA) in the supernatant. Each of identified metabolites accounted for <10% of TECRA. Mobocertinib underwent extensive first-pass metabolism with the fraction of the dose absorbed estimated to be approximately 91.7%. Fecal excretion of mobocertinib metabolites was the major elimination route. Mobocertinib was mainly eliminated via oxidative metabolism with a fraction of approximately 88% metabolized by CYP3A4/5. The other minor elimination pathways included cysteine conjugation, metabolism by other cytochrome P450s, and renal excretion of unchanged mobocertinib. SIGNIFICANCE STATEMENT: This article describes the metabolism and excretion of a targeted covalent inhibitor mobocertinib in humans after a single oral administration of [14C]mobocertinib. Mobocertinib was highly covalently bound to human plasma proteins. No metabolite accounted for >10% of total extracted circulating radioactivity in human plasma. Mobocertinib was mainly eliminated via CYP3A4/5 mediated oxidative metabolism followed by fecal excretion after approximately 91.7% of the dose was absorbed.

Resistance mechanisms of EGFR tyrosine kinase inhibitors, in EGFR exon 20 insertion-mutant lung cancer.

BACKGROUND: Mobocertinib, an EGFR exon 20 insertion (Ex20ins)-specific tyrosine kinase inhibitor has been used for treatment of advanced/metastatic EGFR Ex20ins-mutant non-small cell lung cancer (NSCLC). However, resistance mechanisms to EGFR Ex20ins-specific inhibitors and the efficacy of subsequent amivantamab treatment is unknown. METHODS: To investigate resistance mechanisms, tissue and cfDNA samples were collected before treatment initiation and upon development of resistance from NSCLC patients with EGFR Ex20ins mutations received mobocertinib, poziotinib, and amivantamab treatments. Genetic alterations were analyzed using whole-genome and targeted sequencing, and in vitro resistant cell lines were generated for validation. RESULTS: EGFR amplification (n = 6, including 2 broad copy number gain) and EGFR secondary mutation (n = 3) were observed at the resistance of mobocertinib. One patient had both EGFR secondary mutation and high EGFR focal amplification. In vitro models harboring EGFR alterations were constructed to validate resistance mechanisms and identify overcoming strategies to resistance. Acquired EGFR-dependent alterations were found to mediate resistance to mobocertinib in patients and in vitro models. Furthermore, two of six patients who received sequential amivantamab followed by an EGFR tyrosine kinase inhibitor had MET amplification and showed partial response. CONCLUSIONS: Our study revealed EGFR-dependent and -independent mechanisms of mobocertinib resistance in patients with advanced EGFR Ex20ins-mutant NSCLC.